Amplification of Nucleic Acids

Polymerase Chain Reaction (PCR)

PCR is an in vitro enzymatic reaction used to amplify a defined region of

DNA. It uses:

●​ Two oligonucleotide primers (anneal to 5' and 3' ends of the target
sequence)
●​ DNA polymerase (commonly Taq polymerase, which is
thermostable) (withstand high temp)
●​ dNTPs (deoxyribonucleoside triphosphates) *add more abt this

Mechanism

●​ PCR consists of repeated cycles of:

1.​ Denaturation (94°C): DNA strands separate.
2.​ Annealing (50–60°C): Primers bind to complementary
sequences.
3.​ Extension (72–74°C): Taq polymerase synthesizes new
DNA. Three Steps of a PCR Reaction
●​ Each cycle doubles the number of DNA molecules.
●​ After 30 cycles, over 1 billion copies of the target DNA can be 1.​ Denaturation (94°C)
produced, starting from even a single molecule. ○​ Breaks hydrogen bonds.
●​ Temperature allows to have this cycle ○​ DNA becomes single-stranded.
2.​ Annealing (50–60°C)
Requirements ○​ Primers bind to target sequences.
○​ Temperature is set about 2°C below the melting
●​ Known sequences at the borders of the target DNA region. temperature of the primers. (
●​ Thermostable polymerase (Taq polymerase from Thermus ○​ Primer is magka hiwalay or no?
aquaticus) to withstand high temperatures.​ ○​ Lower temperature to bind to target to sequence (to
ensure)
3.​ Extension (72–74°C)
○​ Taq polymerase extends the primers.
○​ New DNA strands are synthesized.
Product Generation

●​ 1st Cycle: Long products with random 3' ends.

●​ 2nd Cycle: More DNA is synthesized, including two fully new strands.
●​ 3rd Cycle: Correct amplification products appear.
●​ Subsequent cycles: Exponential amplification of the correct product.

Analysis of PCR Products

●​ Agarose Gel Electrophoresis

○​ Detects DNA bands stained with ethidium bromide.
○​ Visual confirmation of amplification success.
●​ Southern Blot Hybridization
○​ Used when DNA yield is low.
●​ Other Techniques
○​ DNA sequencing
○​ Cloning

Interpretation

●​ Expected band present: Successful PCR.

●​ No band or unexpected bands: PCR failed or had errors; may
need repetition.
 Modified Nucleic Acid Amplification Techniques 2. Nested Polymerase Chain Reaction
●​ Increases sensitivity and specificity. (bc of 2 sets of rounds.
1. Reverse-Transcriptase Polymerase Chain Reaction Shorter on second round so more specific)
(RT-PCR) ●​ Uses two sets of primers and two rounds of PCR:
○​ First Round: Amplifies a broad region.
●​ Used to amplify RNA targets. ○​ Second Round: Uses new primers that bind within the
●​ Steps: first PCR product.
○​ Reverse transcription of RNA into cDNA (complementary ●​ Concern: Risk of contamination between rounds.
DNA) using reverse transcriptase. ●​ Solution: Use of wax/oil to separate reaction phases.
○​ Amplification of cDNA by conventional PCR.
●​ Primers Used:
○​ Oligo dT primers: Bind to poly-A tails of mRNA.
○​ Random hexamers/decamers: Bind randomly on RNA to
generate cDNA from all RNA present.

*Product yield is DNA because RNA is fragile

3. Multiplex Polymerase Chain Reaction

●​ Amplifies multiple target sequences in a single PCR reaction.

●​ Applications:
○​ Simultaneous detection of multiple pathogens.
○​ Saves time, effort, and resources.
●​ Requirements:
○​ Primers must have similar annealing temperatures.
○​ No cross-complementarity between primers.
○​ Amplicon sizes must be distinct for gel separation.
 ●​ Types:
○​ Single-template multiplex: One DNA template, multiple
✅ Summary Table: PCR Techniques
primer sets. Technique Target Key Feature Application
○​ Multiple-template multiplex: Multiple templates, multiple
primer sets. Conventional DNA Amplifies specific Detection of specific DNA
PCR DNA region sequences
●​ Advantages:
○​ Internal control reduces false results.
○​ Cost-effective and time-efficient. RT-PCR RNA RNA → cDNA → Gene expression, RNA virus
●​ Disadvantages: Amplification detection
○​ Technically complex.
○​ Often less sensitive than single-primer-set PCR.
Nested PCR DNA Two primer sets, Increased specificity and
2-step amplification sensitivity

Multiplex PCR DNA Multiple targets in Simultaneous detection of

one reaction multiple sequences

Digital PCR DNA/R Absolute Rare mutation detection,

NA quantification (digital low-abundance DNA
read) quantitation

Parameters That Affect PCR

1. Primer Length and Annealing Temperature

4. Digital Polymerase Chain Reaction ●​ The two most important parameters in PCR.
●​ Converts PCR from exponential to digital analysis. ●​ Correct primer design → amplification of a single target DNA
●​ Detects presence or absence of amplification rather than fragment.
quantity. ●​ Incorrect design → no amplification, or nonspecific/multiple
●​ Mechanism: fragments amplified.
○​ Isolates single nucleic acid molecules in
chambers/capillaries/microemulsions/arrays. PCR Product Size
○​ PCR occurs within each isolated zone.
○​ Count of positive zones gives absolute quantification of ●​ First step in primer design: identify the target region.
nucleic acids. ●​ Ideal target sequence: < 3 kb.
●​ Applications: ●​ Up to 10 kb: achievable with standard PCR, but with reduced
○​ Detection of low-level pathogens. efficiency and accuracy.
○​ Identification of rare genetic sequences in single cells. ●​ Long-range PCR (up to 20–40 kb): achieved using:
○​ Clonal amplification for sequencing mixed nucleic acids.​ ○​ A thermostable polymerase, and
 ○​ A second polymerase with 3'→5' exonuclease
(proofreading) activity.
○​ WHY IS 5 BASE PAIRS NOT GOOD? -too short
○​ What is ideal?

Applications: cloning, expression experiments, cDNA analysis.

Primer Length
●​ Short primers (<10-mers) → may hybridize to multiple nontarget
sites → nonspecific products.
○​ Example: In S. cerevisiae DNA, 5-mers (~1024 bp frequency)
result in ~11,788 possible sites in the 12,071,326 bp genome.
●​ Long primers (~17-mers) → very high specificity due to rare
hybridization sites (1 in ~17 billion).
●​ Trade-off:
○​ Long primers → better specificity, but slower hybridization,
and higher annealing temperature. Ideal Annealing Temperature:
○​ Optimal primer length: < 30 nucleotides.
○​ 10k base pairs- time consuming ●​ Should be:
○​ Low enough for correct hybridization.
Annealing Temperature ○​ High enough to avoid mismatched pairing.

PCR Cycle Temperatures: ●​ Determined by the melting temperature (Tm) of the

primer-template hybrid.
1.​ Denaturation – ~94°C → Breaks H-bonds between DNA strands.
2.​ Annealing – ~50°C to 60°C → Primers bind to template DNA. Melting Temperature (Tm)
3.​ Extension – ~72°C to 74°C → DNA synthesis by Taq polymerase. Tm: temperature at which 50% of DNA duplexes denature (i.e., strands
separate).
*hydrogen bonds determine melting temp. Longer sequence more
Key Notes:
Hbonds- higher ™
*Richer G/C content higher melting point (1 G/C = 3 Hbond)
●​ Annealing temperature is the most variable and critical for
PCR success.
●​ Too high → no primer binding (high stringency). General Tm Formula:
●​ Too low → mismatches occur (low stringency).
Where: qPCR vs Conventional PCR
●​ M: [KCl] ~25–50 mM
●​ %GC: G/C content in primer Feature qPCR Conventional PCR
●​ %form: formaldehyde %
●​ L: primer length Sensitivity High Semi-quantitative

Simplified Formula: Output Quantitative or Qualitative only

qualitative
Data Real-time (during cycles) Post-PCR analysis only
Collection
●​ Primers must have similar Tm for efficient amplification. Contamination Lower (no post-PCR Higher (due to gel
●​ GC content ↑ → Tm ↑ (G-C has 3 H-bonds, A-T has 2). Risk processing) electrophoresis)

Quantitative PCR (qPCR or Real-Time PCR)

qPCR Instrumentation
●​ qPCR quantifies DNA as it is being amplified (real time).
●​ Thermal cycler
●​ Detects amplicon accumulation after each cycle.
●​ Excitation light source (lamp, laser, or LED)
●​ Compares amplification of target with internal control (known
●​ Fluorescence detector
copy number).
●​ Software to visualize amplification curves

Applications: qPCR Detection Chemistry

1. DNA Binding Dye (Non-Specific)
●​ Gene expression analysis ●​ Example: SYBR Green
●​ Routine DNA quantification ●​ Binds to any dsDNA
●​ Pathogen detection ●​ Pros:
●​ Mutation/SNP analysis ○​ Cost-effective
●​ MicroRNA and methylated DNA analysis ○​ Compatible with multiple primers
●​ Forensic studies ●​ Cons:
●​ GMO quantification ○​ Detects both specific and nonspecific products
●​ Drug efficacy validation
2. Sequence-Specific Fluorescent Probes
●​ Bind only to target amplicon
●​ Pros:
○​ High specificity
○​ Enables multiplexing (multiple gene targets with different
dyes)
 Cycle Threshold (CT) ​

●​ CT: the cycle number where fluorescence exceeds background.

●​ In log phase, CT is inversely proportional to starting copy ●​ SYBR Green:
number. ○​ A highly sensitive asymmetrical cyanine dye.
○​ Fewer cycles → higher initial concentration. ○​ Chemical Name:
2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihyd
ro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl
Summary of PCR Best Practices quinolinium.
○​ Has two positive charges, contributing to high dsDNA
Parameter Best Practice binding affinity.
Primer Length ~18–30 nucleotides
○​ Absorption & Emission: Absorbs at λmax = 497 nm,
emits green light at λmax = 520 nm.
○​ Compatible with most qPCR instruments.
Primer Tm 1–2°C above annealing temp, similar for both primers

Product Size <3 kb ideal, up to 40 kb possible with enhanced enzymes

Annealing Optimized per Tm; high enough for specificity, low enough for
Temperature hybridization

Polymerase Choice Use proofreading enzyme for long-range, high-fidelity

amplification

Principles of Intercalating Dyes

qPCR Detection Choose based on specificity and budget (dye vs probe-based)
●​ Intercalate into the minor groove of any dsDNA, increasing fluorescence.
●​ Fluorescence is measured during the extension phase of each PCR
cycle.
DNA INTERCALATING DYES FOR qPCR ●​ Non-specific binding may cause primer-dimer formation or nonspecific
products.​
Early Dyes in Real-Time PCR
●​ Melting Curve Analysis:
●​ Ethidium Bromide: First used by directly adding it to the PCR ○​ Run from 50°C to 95°C to check product specificity.
mix and monitoring fluorescence. ○​ DNA denaturation = sharp drop in fluorescence.
○​ Specific products melt at higher temperatures; nonspecific
products show multiple peaks.
○​ Useful in distinguishing specific vs nonspecific amplicons.
 Comparison: SYBR Green vs EvaGreen Types of Fluorophores
Property SYBR Green EvaGreen ●​ Donor/Reporter: Absorbs light, becomes excited, emits
lower-energy fluorescence.
PCR Inhibition Inhibitory at >0.5 μM Low inhibition, usable at 1.34 ●​ Acceptor/Quencher: Accepts energy via FRET (Fluorescence
μM
Resonance Energy Transfer) when close to donor.
●​ FRET only occurs within 10–100 Å distance.
Stability Relatively unstable in alkaline Very stable ●​ Separation during amplification allows fluorescence emission.
pH ●​ Fluorescent oligonucleotides:
HRM Poor due to dye redistribution Excellent, suitable for ○​ Primer-probes
Compatibility closed-tube HRM ○​ Probes
○​ Nucleic acid analogues
Signal Strength Weaker, may affect melt curve Stronger signal, better melt
accuracy curve Primer-Probe Chemistries
Hairpin Primer-Probes
Mutagenicity Weakly mutagenic but may Lower toxicity profile Type Key Features & Fluorescence Mechanism
enhance mutation
Scorpions - Hairpin with reporter (5’) and quencher (3’)
Multiplex Use Limited More suitable
●​ HEG linker blocks primer extension
Fast Cycling Not ideal Compatible with fast cycling
protocols ●​ FRET quenching when intact
●​ Fluorescence emitted upon hairpin opening
●​ Used in single/multiplex qPCR for genotyping, SNP, mutation
detection | | Amplifluor | - Similar to Scorpions
FLUOROPHORE-LABELED OLIGONUCLEOTIDES FOR ●​ Reporter (5’), quencher (3’)
qPCR ●​ Fluorescence increases after denaturation
●​ Applications: SNP, genotyping, pathogen/GMO detection | | LUX |
Principle - No quencher; uses G-rich 3’ region
●​ Fluorescence up to 8× after extension
●​ Use fluorescently labeled primers (fluorophores) as probes. ●​ Used in expression analysis, SNP/genotyping, GMO detection |
●​ More specific and reliable than intercalating dyes.
●​ No free dye needed; fluorescence is from labeled
oligonucleotides.
●​ Simple assay design.
Cyclicon Primer-Probes Summary Table of Primer-Probe Systems
●​ Composed of:
○​ Long primer-probe complementary to target System Structure Detection Needs Application Notes
○​ Short oligo bound via 5’-5’ ends Type Phase Quencher
○​ Quencher on thymine at 5’ position​
Scorpion Hairpin Extension Yes High specificity, minimal
background
●​ Cyclic structure: Maintains quencher proximity pre-binding.
●​ When target is present:
○​ Cyclic structure opens Amplifluor Hairpin Denaturation Yes Multiplex capable
○​ 3’ end of probe is extended
○​ Fluorescence is emitted (FRET is disrupted). LUX Hairpin Extension No Up to 8× signal increase
●​ Advantages:
○​ Cost-effective Cyclicon Cyclic Extension Yes Less contamination, short
○​ Shorter oligos oligos
○​ Reduced background noise
●​ Can also act like TaqMan or Molecular Beacons when
connected 3’-3’. Angler Linear + Denaturation Yes + SYBR No melting needed, dual
SYBR Gold detection

Angler Primer-Probes
●​ Probe sequence matches target DNA
●​ Linked to reverse primer via HEG linker Fluorophore-Labeled Oligonucleotides:
●​ Acceptor fluorophore on 5’ end PROBES-BASED CHEMISTRY
●​ Uses SYBR Gold as donor (intercalating dye)
●​ In solution: No fluorescence (no FRET) Fluorophore-labeled oligonucleotides are short, synthetic DNA probes
●​ During PCR: used to increase the specificity of PCR reactions. These probes contain:
○​ Probe binds, extension occurs
○​ Denaturation causes probe hybridization ●​ A reporter fluorophore (usually at the 5’ end)
○​ SYBR Gold intercalates → Fluorescence ●​ A quencher (usually at the 3’ end)
●​ Detects both specific (Angler) and nonspecific (SYBR Gold)
products without melting curves The fluorescence signal is quenched when both dyes are in close
●​ Applications: proximity. Signal is released (fluorescence detected) only when the
○​ Rapid DNA detection probe is degraded or unfolded, separating the dyes.
○​ Gene expression studies
○​ SNP and mutation detection There are two main types of probes:
○​ Environmental/biological sample screening
1.​ Hydrolysis Probes
2.​ Hybridization Probes
Hydrolysis Probes ●​ Provides higher Tm and reduces non-specific amplification.
●​ FRET quenching occurs when the probe is in random coil form.
●​ Binding straightens the probe, increasing fluorescence.
✳ TaqMan Probes (Prototype Hydrolysis Probe)
Applications:
●​ Use the 5’→3’ exonuclease activity of Taq polymerase to
degrade the probe. Same as TaqMan, plus:
●​ Fluorescence is measured at the end of the extension phase. ●​ Forensic analysis
●​ Fluorescence is proportional to the amount of specific
product. Hybridization Probes
Mechanism: ●​ Fluorescence detected during annealing or extension phase
●​ Allow melting curve analysis
1.​ The probe binds to a specific region of the target DNA between ●​ Fluorescence is directly proportional to the amplified product
two PCR primers.
2.​ In solution, the fluorophore’s signal is quenched by the acceptor. ✳ HybProbes (FRET Probes)
3.​ During extension, Taq polymerase degrades the probe, ●​ Use two adjacent oligonucleotide probes:
separating the fluorophore from the quencher. ○​ One with reporter fluorophore at 3’ end
4.​ Fluorescence increases. ○​ One with quencher at 5’ end + 3’ phosphate to block
extension
Applications: ●​ Probes hybridize in a head-to-tail orientation.

●​ Single & multiplex qPCR Mechanism:

●​ Virus detection 1.​ During annealing, probes bind adjacently.
●​ Viral/bacterial load quantitation 2.​ Fluorescence occurs when quencher is excited by energy from the
●​ Gene expression reporter.
●​ Microarray validation Advantages:
●​ Allelic discrimination ●​ Easy design and synthesis
●​ Mutation detection ●​ Quick PCR optimization
●​ SNP detection
●​ GMO detection Applications:

🔹 Minor Groove Binder (MGB) Probes ●​ Pathogen detection

●​ Load quantitation
●​ Microarray validation
●​ TaqMan probes conjugated with an MGB ligand to increase ●​ Genotyping
specificity and sensitivity. ●​ Allelic discrimination
●​ MGB: Small tripeptide that binds noncovalently to the minor ●​ Mutation detection
groove of dsDNA, especially AT-rich regions. ●​ SNP detection
✳ Molecular Beacons Locked Nucleic Acids (LNAs)
●​ Hairpin-shaped, single-stranded probes ●​ Modified nucleotides with a bicyclic methylene bridge between
●​ Consist of: 2’-O and 4’-C on the ribose
○​ Loop (18–30 bp) complementary to target ●​ Introduced into Molecular Beacon or TaqMan probes
○​ Stem (5–7 bp inverted repeats)
Mechanism: Properties:
1.​ In closed form, fluorophore & quencher are close → no ●​ Resist degradation
fluorescence. ●​ Increase probe stability and Tm
2.​ During annealing, beacon binds target and unfolds → ●​ High sequence specificity
fluorescence emitted.
3.​ If not perfectly complementary → remains closed → no signal. Zip Nucleic Acids (ZNAs)
Advantages:
●​ High specificity ●​ Oligonucleotides with cationic Z units (e.g., spermine derivatives)
●​ Can discriminate single-nucleotide differences ●​ Increase attraction to nucleic acids during annealing
Applications:
●​ Pathogen detection Mechanism:
●​ Load quantitation 1.​ Z units are attracted to nucleic acids.
●​ Genotyping 2.​ Scanning begins.
●​ Allelic/SNP discrimination 3.​ Complementary sequence is found → "zipping up" occurs.
●​ Mutation detection
●​ mRNA analysis in living cells Advantage:
●​ GMO detection ●​ Exceptionally high affinity due to polycationic interaction

Nucleic Acid Analogues Nonnatural Bases: Plexor Primers

Nucleic acid analogues resemble DNA/RNA but offer greater stability and ●​ Use of modified bases:
affinity in biological conditions. ○​ Isoguanine (Iso-dG)
○​ 5-Methylisocytosine (Iso-dC)
Peptide Nucleic Acids (PNAs)
●​ Sugar-phosphate backbone is replaced with Mechanism:
N-(2-aminoethyl)-glycine peptide 1.​ One primer has Iso-dC and a fluorescent reporter at 5’ end.
●​ Electrically neutral 2.​ Iso-dG nucleotides, attached to a quencher, are included in the
●​ Bind via strand displacement (not triple helix) reaction.
●​ Attach to fluorophores or thiazole orange for qPCR 3.​ As amplification proceeds, quencher and reporter get close →
Properties: fluorescence is quenched.
●​ High specificity and affinity 4.​ Decrease in fluorescence is proportional to the initial amount of
●​ Resistant to nucleases & proteases target DNA.​
●​ Work well in low salt concentrations
 Summary Table ISOTHERMAL AMPLIFICATION TECHNIQUES
Type Mechanism Key Feature Can Do Applications One major limitation of PCR in molecular diagnostics is the need for a
Melting
Curve? thermocycler to denature DNA strands—equipment that is expensive and
not portable. To overcome this, isothermal amplification techniques were
developed to enable nucleic acid amplification at a constant temperature
TaqMan Hydrolysis 5'→3'
exonuclease
❌ Quantification,
mutation detection,
without thermocycling. These methods are based on the molecular biology
of DNA/RNA synthesis using polymerases and auxiliary proteins under
activity etc. isothermal conditions.

1.​ Nucleic Acid Sequence-Based Amplification (NASBA)

MGB Probes Hydrolysis + Increased Tm, ❌ High sensitivity ●​ Transcription-based amplification: Includes NASBA and TMA
groove binding AT-rich binding assays (Transcription-Mediated Amplification).​

●​ Mimics retroviral RNA replication: Targets RNA sequences

specifically.
HybProbe Hybridization FRET between
adjacent probes
✅ Multiplex detection
●​ Enzymes involved:
○​ Reverse transcriptase: Converts RNA to cDNA.
○​ RNase H (in NASBA): Degrades RNA in RNA-DNA
hybrids.
Molecular
Beacon
Hairpin
hybridization
High specificity ✅ SNP/mutation
detection
○​ T7 RNA polymerase: Synthesizes RNA from dsDNA
containing T7 promoter.
●​ Mechanism:
PNA Strand
displacement
Nuclease
resistance
❌ Highly stable
assays
○​ Forward primer binds target RNA.
○​ RT and RNase H generate dsDNA with T7 promoter.
○​ T7 RNA polymerase synthesizes many RNA copies.
LNA Locked ribose High Tm &
specificity
❌ Used with
TaqMan/Beacons
○​ Amplification is exponential (self-sustained sequence
replication).
●​ Detection: Gel electrophoresis, fluorescent probes, colorimetric

❌
assay.
ZNA Polycationic High affinity Efficient target
binding scanning

Plexor Quenching by
proximity
Fluorescence
decrease
❌ Quantification by
signal loss
2.​ Signal-Mediated Amplification of RNA Technology 4.​ Rolling Circle Amplification (RCA)
(SMART)
●​ Template: Circular DNA.
●​ Based on formation of a Three-Way Junction (3WJ) structure. ●​ Enzyme: Φ29 DNA polymerase.
●​ Probes: Two single-stranded oligonucleotide probes hybridize ●​ Mechanism:​
adjacent to the target and to each other.
●​ Steps: ○​ Primer hybridizes to circle.
○​ Bst DNA polymerase extends short probe to form T7 ○​ Polymerase continuously replicates template, displacing
promoter. previous strands.
○​ T7 RNA polymerase generates RNA amplicons. ●​ Product: Long ssDNA with tandem repeats.
○​ Secondary amplification using additional probes. ●​ Applications: Detection of DNA/RNA, proteins, biomarkers.
●​ Target-dependent signal: Detection via enzyme-linked
oligosorbent assay or real-time methods. 5.​ Loop-Mediated Isothermal Amplification (LAMP)
●​ Does not copy target sequences: It amplifies signal only.​
●​ Primers: Four primers recognize six regions of target DNA.
3.​ Strand Displacement Amplification (SDA) ●​ Polymerase: With strand displacement activity.
●​ Conditions: Constant temp (~60°C), time <1 hr.
●​ Enzymes used: ●​ Product: Stem-loop DNA with cauliflower-like structures.
○​ Restriction endonuclease (e.g., HincII): Nicks DNA at ●​ Advantages:
specific sites. ○​ No thermocycler needed.
○​ Exonuclease-deficient DNA polymerase (e.g., exo– ○​ Amplifies DNA and RNA (with RT).
Klenow): Strand displacement activity. ○​ High specificity and sensitivity.
●​ Process: ○​ Detection via turbidity (magnesium pyrophosphate) or
○​ Initial heat denaturation. fluorescence.
○​ Four primers (B1, B2, S1, S2) amplify target DNA with ●​ Applications: POCT, genetic testing, pathogen detection in
nicking sites. clinical/food/environmental samples.
○​ HincII and polymerase generate displaced strands,
entering continuous nick-extension-displacement cycles. 6.​ Isothermal Multiple Displacement Amplification (IMDA)
●​ Amplification: Exponential (~10¹⁰-fold).
●​ Application: Chlamydia trachomatis and Neisseria gonorrhoeae ●​ Principle: Strand displacement using multiple primers.
detection. ●​ Primers: Two sets (left & right) for opposite DNA strands.
●​ Process:
○​ Primer hybridization initiates replication.
○​ Strand displacement amplifies nucleic acid.
●​ Applications: Whole genome amplification, especially for limited
samples.
7.​ Single Primer Isothermal Amplification (SPIA) Circular HDA (cHDA)
●​ Template: Circular DNA.
a. DNA Amplification ●​ System: T7 replication machinery (T7 helicase +
exonuclease-deficient T7 polymerase).
●​ Primer: Chimeric (3’ DNA + 5’ RNA). ●​ Mechanism:
●​ Enzymes: ○​ Primer binds circular ssDNA.
○​ RNase H: Cleaves RNA in hybrids. ○​ Polymerase replicates & displaces newly synthesized
○​ DNA polymerase with strand displacement activity. strands.
●​ Process: ○​ Rolling-circle amplification generates long DNA
○​ Primer binds to target. concatemers.
○​ Extended product displaced by new primer. ●​ Detection: Gel electrophoresis, qPCR, ELISA.​
○​ Cycle repeats for exponential amplification.

b. RNA Amplification (Ribo-SPIA) Central Dogma Theory of Molecular Biology and

Genetic Code
●​ Application: Amplification of total or specific RNA.
●​ Mechanism:
○​ Linear isothermal amplification. What is the Central Dogma of Molecular Biology?
○​ Up to 10,000-fold replication of each original transcript.
●​ Use: Useful in clinical research with low-yield samples.​ The Central Dogma of molecular biology, a pivotal concept in modern
biology, delineates the flow of genetic information within a biological
8.​ Helicase-Dependent Amplification (HDA) system. This concept, first elucidated by Francis Crick in 1957, posits a
unidirectional information flow from DNA to RNA, culminating in the
●​ Principle: Mimics DNA replication fork. synthesis of proteins. It has profoundly influenced our understanding of
●​ Enzymes: genetic information storage, replication, and expression in living
1.​ DNA helicase: Separates dsDNA. organisms.
2.​ Single-stranded binding proteins (SSBs): Stabilize ssDNA.
3.​ DNA polymerase: Synthesizes new DNA strands. Central Dogma Steps
●​ Temperature: Constant (60–65°C), no heat denaturation.
●​ Steps: 1.​ DNA Replication: This initial step involves the precise duplication
1.​ Helicase unwinds DNA. of DNA molecules. Occurring during cell division, it ensures the
2.​ Primers bind. transfer of identical genetic information to daughter cells. DNA
3.​ Polymerase extends primers. replication is fundamental in maintaining the integrity of genetic
4.​ New dsDNA serves as template. information across generations.
●​ Amplification: Exponential via chain reaction.
●​ Applications: HIV-1, Clostridium difficile detection. ​
 2.​ Transcription: In this stage, a specific DNA segment serves as a Relation between Central Dogma and Genetic Code
template for synthesizing a complementary RNA molecule,
particularly messenger RNA (mRNA). This process translates the The Central Dogma describes the flow of genetic information from DNA to
genetic code from DNA to RNA, facilitating its transport to the RNA to protein, while the genetic code provides the language in which this
cell’s cytoplasm.​ information is translated. During transcription, DNA is transcribed into
mRNA, which carries the genetic code. During translation, this code is
3.​ Translation: The final stage of the Central Dogma, translation, read in sets of three nucleotides (codons), each specifying a particular
involves the synthesis of proteins. Here, the genetic code borne by amino acid, leading to the synthesis of proteins.
mRNA directs the assembly of amino acids into proteins. This
process occurs in ribosomes, where transfer RNA (tRNA)
Exceptions to the Central Dogma
molecules bring amino acids in the sequence specified by the
While the Central Dogma outlines a general flow of genetic information,
mRNA.
there are notable exceptions:
Therefore, the Central Dogma encapsulates a sequential and systematic
●​ Reverse Transcription: Certain viruses, like retroviruses, can
flow of genetic information, starting from DNA replication, through
reverse-transcribe their RNA into DNA, integrating it into the host
transcription to mRNA, and culminating in protein synthesis during
genome.
translation. It’s crucial to recognize that this model, while generally
●​ RNA Replication: Some RNA viruses replicate their RNA
applicable, has its exceptions and complexities, such as
genomes without a DNA intermediate.
post-transcriptional modifications and the roles of non-coding RNAs in
●​ Direct DNA to Protein: In vitro experiments have demonstrated
gene regulation.
the possibility of synthesizing proteins directly from DNA without
an RNA intermediate, though this is not observed in natural
What is Genetic Code? biological systems.
The genetic code is the set of rules by which information encoded within
genetic material (DNA or RNA sequences) is translated into proteins by General Transfers of Biological Sequential Information
living cells. Specifically, it defines how sequences of nucleotide triplets,
These are the standard processes observed in most cells:
called codons, specify which amino acid will be added next during protein
●​ DNA to DNA: Replication.
synthesis.
●​ DNA to RNA: Transcription.
●​ RNA to Protein: Translation.​
Key Features of the Genetic Code
●​ Universality: The genetic code is nearly universal, with few
exceptions.
●​ Redundancy: Multiple codons can code for the same amino acid.
●​ Specificity: Each codon specifies only one amino acid.
●​ Non-overlapping: Codons are read one after another without
overlapping.
●​ Start and Stop Signals: Specific codons signal the start and end
of protein synthesis.
Special Transfers of Biological Sequential Information
These occur under specific conditions, often in viruses:

●​ RNA to DNA: Reverse transcription.

●​ RNA to RNA: RNA replication in some viruses.
●​ DNA to Protein: Direct translation from DNA to protein in vitro.

Is the Central Dogma of Biology Unidirectional?

While the traditional view of the Central Dogma suggests a unidirectional
flow of information (DNA → RNA → Protein), the existence of processes
like reverse transcription indicates that, under certain circumstances,
information can flow in reverse (RNA → DNA). However, the flow from
protein back to nucleic acids does not occur, maintaining a general
directionality in the system.

Significance of the Central Dogma of Molecular Biology

Understanding the Central Dogma is crucial for comprehending how
genetic information is stored, expressed, and transmitted in living
organisms. It underpins many areas of biology and medicine, including
genetic engineering, molecular diagnostics, and the development of
therapies for genetic diseases.

What is the Central Dogma of molecular biology?

The Central Dogma is a concept that describes the flow of genetic
information within a biological system, specifically the transfer of
information from DNA to RNA to protein.

What are the three general transfers in the Central Dogma?

●​ DNA to DNA (Replication)
●​ DNA to RNA (Transcription)
●​ RNA to Protein (Translation)

What are the three special transfers in the Central Dogma?

●​ RNA to DNA (Reverse Transcription)
●​ RNA to RNA​
 Techniques in DNA Sequencing DNA Polymerase Used
●​ Klenow Fragment: Low processivity, yields ~250 bp per reaction.
CHAIN-TERMINATION DNA SEQUENCING (Sanger ●​ Sequenase (T7 DNA Polymerase Variant):​
Sequencing)
○​ High processivity
Principle of Dideoxynucleotide Procedure ○​ No exonuclease activity
○​ Sequences up to ~750 bp per reaction.
●​ Also called Sanger Sequencing.
●​ Relies on DNA synthesis requiring a free 3'-OH group on the
sugar of the last nucleotide. Template Preparation
●​ If a dideoxynucleotide (ddNTP) is incorporated (lacking a 3'-OH), ●​ Must be single-stranded DNA:
synthesis terminates—no phosphodiester bond can form. ○​ M13 Vector (≤3 kb inserts)
●​ This stops elongation, forming DNA fragments of varying ○​ Plasmids (requires denaturation—alkali/boiling)
lengths. ○​ Phagemids (plasmids w/ M13 origin) → support both
single- and double-stranded forms; accommodate larger
Procedure Overview fragments (~10 kb+)
1.​ Template: Identical single-stranded DNA molecules.
2.​ Primer: A short oligonucleotide (17–24 nucleotides) annealed to a
AUTOMATED DNA SEQUENCING
specific site on the template.
3.​ DNA Polymerase: Originally Klenow fragment; synthesizes the
complementary strand. ●​ Built on Sanger method, but uses fluorescent-labeled
4.​ Reaction Setup: 4 tubes, each with: ddNTPs (instead of radioactive dATP).
○​ All 4 dNTPs (dATP, dTTP, dCTP, dGTP) ●​ Products separated via:
○​ One specific ddNTP (e.g., ddATP, ddTTP, ddCTP, ddGTP) ○​ Polyacrylamide gel electrophoresis or
5.​ Termination: ddNTPs randomly terminate synthesis, generating ○​ Capillary electrophoresis
fragment pools ending in A, T, C, or G. ●​ Fluorescent detector reads the label → identifies the
6.​ Separation: Gel electrophoresis under denaturing conditions. terminal base (A, T, C, G).
7.​ Visualization: Radioactive dATP enables autoradiography → ●​ Output: Printed sequence or saved to a digital database.
DNA bands on X-ray film = readable sequence.​
●​ Greatly increased sequencing speed → vital for genome
sequencing projects.​
FIRST HIGH-THROUGHPUT TECHNIQUE: Comparison Snapshot
PYROSEQUENCING
Feature Chain-Termination (Sanger) Pyrosequencing
●​ Detects pyrophosphate (PPi) released during DNA
polymerization. Reaction Type Terminates via ddNTP Real-time detection via
incorporation PPi
●​ Steps:
1.​ Start with single-stranded DNA (often via PCR product
denaturation). Output per Reaction ~750 bp max (with ~150 bp per reaction
2.​ Add a primer and use DNA polymerase (commonly Sequenase)
Klenow).
3.​ When a dNTP is added: Parallelization Limited Highly parallel (NGS)
■​ PPi is released.
■​ ATP sulfurylase converts PPi + APS → ATP. Detection Method Radioactive or fluorescent Light emission
■​ Luciferase uses ATP to convert luciferin → label
oxyluciferin, releasing light.
Electrophoresis Yes No
4.​ Light intensity ∝ number of nucleotides incorporated.
Required?

Key Steps
Speed Slower Faster
●​ Nucleotides added one at a time.
●​ If incorporated → flash of light. Ideal For Smaller-scale, accurate Large-scale,
●​ If not → apyrase degrades unincorporated dNTPs before next is sequencing high-throughput
added.
●​ No electrophoresis required → faster than Sanger sequencing.

Advantages of Pyrosequencing

●​ Can be massively parallelized (many reactions at once).

●​ Output up to 1000 million base pairs (1 Gb) per run.
●​ Although it only produces sequences of up to 150 bp, it's very
fast and ideal for large-scale sequencing.
Techniques to Sequence a Genome Limitations

●​ Genome sequencing reveals the genetic blueprint of organisms. ●​ Works best for small bacterial genomes with few repetitive
●​ It is crucial in understanding organism biology, human elements.
diseases, and pathogen identification. ●​ Not ideal for eukaryotic genomes due to repeat sequences
●​ The first sequenced DNA genome was bacteriophage φX174 causing assembly errors.​
(5375 bp), followed by SV40 virus, pBR322, and eventually
human mitochondrial genome and bacteriophage λ.
2. Clone Contig Approach
Genome Size Overview
●​ More suitable for large or repetitive genomes.
●​ Requires pre-sequencing mapping of overlapping clones
Organism Type Genome Size
(contigs).
●​ Uses BACs or YACs to handle large fragments (up to 1.5 Mb).
Typical Bacterial ~4,000,000 bp
Assembly via Overlap
Human Genome ~3,200,000,000 bp
●​ Clones are sequenced from both ends (producing pair ends,
Pyrosequencing Output Up to 150 bp/read 600–700 bp).
●​ Shared sequences are clustered and assembled into the genome
Challenge: Assembling millions of short reads into a complete sequence. step-by-step.
●​ Long inserts reduce the number of clones needed.
Two Main Sequencing Strategies
Clone Contig Assembly Techniques
1. Shotgun Sequencing
●​ Random fragmentation of genomic DNA. A. Chromosome Walking
●​ DNA fragments are inserted into universal vectors, forming a
shotgun library.​ ●​ Step-by-step assembly from a starting clone.
Primers target vector flanking regions to sequence both fragment ●​ Uses probes to detect overlapping clones via hybridization.
ends (via dideoxynucleotide method). ●​ Slowly builds a contig by repeating hybridization with new
overlapping clones.
Overlap Assembly
●​ Sequence fragments are compared for overlaps. Limitations
●​ The goal is to assemble contiguous sequences (contigs).
●​ Accuracy is critical; misalignment leads to scrambled or ●​ Time-consuming and labor-intensive.
incomplete genomes. ●​ Risk of false positives from repeat sequences in probes.
 B. Rapid Clone Fingerprinting Methods Fingerprinting Rapid, no start point Requires multiple assays
Methods needed
●​ Don’t require a fixed start point; faster than chromosome walking.​
Aim to identify pairs of overlapping clones. STS Analysis Highly specific, maps Requires prior knowledge
clones precisely of STS
1. Restriction Fragment Fingerprinting
●​ Digest clones with restriction enzymes.
●​ Run on agarose gel.
●​ Clones with shared bands (excluding vector bands) likely overlap.
High-Throughput NGS Strategies
2. Repetitive DNA PCR / IRE-PCR ●​ Traditional Methods (Shotgun & Clone Contig): Effective but
●​ Uses primers targeting repetitive elements. expensive and time-consuming for large genomes.
●​ Amplifies DNA between adjacent repeats. ●​ High-Throughput NGS: Developed to reduce time, cost, and
●​ Similar band sizes across clones indicate potential overlaps. reagent use by generating dense DNA fragment libraries directly
on solid surfaces or in picoliter wells.
C. Sequence Tagged Site (STS) Content Analysis ●​ Massive Parallelism: Allows sequencing of hundreds of millions
of DNA fragments simultaneously.
●​ Searches for clones containing a unique DNA marker (STS).
●​ STS = sequence tagged site, a DNA sequence that occurs only NGS: General Principles
once in the genome. ●​ Massively Parallel Sequencing: Conducted in a flow cell using
●​ If two clones contain the same STS, they must overlap. either clonally amplified or single DNA molecules.
●​ PCR primers for known STSs are used to probe the library. ●​ Sequencing Approaches:
○​ Sequencing-by-Synthesis (SBS)
○​ Sequencing-by-Ligation
Summary of Strategies ○​ Pyrosequencing
Strategy Advantages Limitations ○​ Semiconductor sequencing

Shotgun Sequencing Fast for small genomes; Ineffective for

automated large/repetitive genomes

Clone Contig Accurate with Time-consuming; requires

Approach large/repetitive genomes mapping

Chromosome Walking Useful for filling gaps Slow, laborious

 2. Illumina/Solexa – Sequencing-by-Synthesis
NGS Platforms Overview
●​ Introduced: 2006
Platform Method Amplification Read Output Key Feature
Length ●​ Core Components: Reversible terminator nucleotides + DNA
polymerase.
●​ Flow Cell: Glass slide with 8 lanes; DNA is immobilized via
Roche 454 Pyrosequencing Emulsion PCR ~700 bp ~500 Long reads; good adapter anchors.
GS FLX Mb/run for de novo
assembly ●​ Amplification: Bridge Amplification creates clusters (polonies)
of ~1000 copies each.
●​ Sequencing Cycle:
○​ Four labeled dNTPs are added → one base incorporated
Illumina Sequencing-by-Sy Bridge 75–300 >1–3 High throughput,
(GA/HiSeq) nthesis Amplification bp Gb/run accurate, low cost → fluorescence recorded.
○​ Blocking group and dye are cleaved → next base can be
added.
SOLiD (AB) Sequencing-by-Lig Emulsion PCR ~35 bp ~4 Dual interrogation ○​ Output: 1.5–3 Gb per run; read lengths: 36–100 bp.
ation Gb/run increases
accuracy (99.9%) ○​ Advantages: High accuracy, low cost, widely used.

3. SOLiD (Supported Oligonucleotide Ligation and

Ion PGM (Ion
Torrent)
Semiconductor
Sequencing
Emulsion PCR ~200 bp Fast runs Detects H⁺
release via pH
Detection) – Sequencing-by-Ligation
changes
●​ Mechanism: Ligation of octamer probes instead of polymerase
extension.
●​ Process:
Platform-Specific Details ○​ Octamers have specific fluorescent tags for dinucleotide
combinations.
1. Roche/454 Life Sciences – Pyrosequencing ○​ Template DNA is hybridized to primers, ligation occurs.
●​ Year Introduced: 2005 (GS FLX in 2007) ○​ Fluorescence read, dye cleaved, next cycle begins.
●​ Core Technology: Emulsion PCR + Pyrosequencing on picotiter ●​ Repeated with 5 different primers, each offset by one
well plates. nucleotide.​
●​ Signal Detection: Light emitted upon incorporation of nucleotide Key Advantage: Each base read twice, increasing accuracy
(via pyrophosphate detection). (99.9%).​
●​ Key Steps: Output: ~4 Gb in 6 days; read length: 35 bases.​
○​ DNA fragmented, ligated to adapters, captured on beads.
○​ Beads undergo emulsion PCR in oil droplets.
○​ Beads placed in wells on a fiber-optic plate.
○​ Each dNTP added in cycles; incorporation → light
emission → CCD camera captures sequence.
4. Ion PGM (Ion Torrent) – Semiconductor Sequencing Key Takeaways

●​ Launched: 2010 ●​ NGS transformed genomics by enabling high-throughput,

●​ Unique Feature: No optics! Uses pH detection to identify low-cost, and accurate DNA sequencing.
nucleotide incorporation. ●​ Different platforms use distinct sequencing chemistries but
●​ How it works: share the goal of massively parallel sequencing.
○​ Polymerase adds nucleotide → releases H⁺ ion → pH ●​ Platform choice depends on application needs: read length,
change detected by semiconductor. accuracy, speed, and data output.
○​ If correct nucleotide → voltage change recorded.
●​ Strengths: Fast, small machine size, suitable for clinical/small
labs. Third-Generation Sequencing (TGS)
●​ Limitations: Lower data throughput than other platforms.
●​ TGS Characteristics:
○​ No PCR required before sequencing → Faster sample
Comparison Table of Key Specs prep
Platform Read Run Output / Notes ○​ Real-time signal capture during enzymatic nucleotide
Length Time Run addition
(bp) ●​ Single-molecule sequencing: Core of TGS; reduces sample
mass requirements, avoids DNA amplification
Roche 454 ~700 Variabl ~500 Mb Longest read length; high
e cost
Advantages:
Illumina 75–300 2–4 1–3 Gb Most popular; high accuracy;
HiSeq days low cost ●​ Allows re-sequencing of the same molecule for better accuracy
●​ Long read lengths and quantitative precision
●​ Suitable for GC-rich, structurally complex DNA, or degraded
SOLiD ~35 ~6 ~4 Gb Extremely accurate; complex samples
days workflow

Ion Torrent ~200 Few ~100s Rapid turnaround; real-time

PGM hours Mb sequencing via pH
Common Third-Generation Sequencing Technologies 3. Life Technologies Method
1. Helicos Single-Molecule Sequencing (HeliScope) ●​ Uses FRET-based sequencing-by-synthesis
●​ Polymerase tagged with quantum dots, interacting with
●​ Sequencing-by-synthesis on a glass flow cell dye-labeled nucleotides
●​ DNA hybridized to surface-anchored primers; polymerase and ●​ Fluorescence shift between quantum dot and nucleotide dye
labeled nucleotides added used to detect base
●​ Fluorescent “Virtual Terminator” nucleotides prevent premature ●​ Can reprime DNA strands for accuracy improvements
incorporation
Applications:
●​ Images taken every cycle → ~120 cycles/run
●​ Throughput: ~1 billion molecules at once, 50 samples/run ●​ Genome assembly
●​ Read length: ~35 nt; Error rate: ~3–5% (mostly deletions) ●​ Structural variation
●​ 30× coverage produces reliable consensus sequences ●​ Haplotyping
●​ Can sequence RNA directly, improving accuracy in expression ●​ Metagenomics
analysis
●​ Ideal for: 4. Nanopore Sequencing
○​ ChIP, RNA expression, copy number variation ●​ Involves threading ssDNA through a nanopore protein (e.g.,
○​ Degraded DNA or low quantity samples α-hemolysin) in a lipid bilayer
●​ Ionic current disruption read as nucleotides pass through
2. Pacific Biosciences (PacBio) Advantages:
●​ Uses zero-mode waveguides (ZMWs) and SMRTbell templates ●​ Long reads (>5 kbp), ultra-fast speed (1 bp/ns)
(circular DNA) ●​ No fluorescent tags required
●​ Fluorescent nucleotides incorporated one-by-one and detected ●​ Works across varying temperatures
real-time ●​ DNA sequenced via depolymerization, minimizing prep time
●​ Creates a “movie” of incorporation events with time-based signal
analysis
●​ Useful for epigenetic studies (e.g., DNA methylation)
Limitations:
●​ Direct RNA sequencing problematic (false insertions from
reverse transcriptase)
●​ Low read count limits transcriptome profiling
Advantages:
●​ Fast prep (4–6 hrs), no PCR → less bias
●​ Long average read length: ~1300 bp
●​ Run time: <1 day
●​ Useful for:
○​ Genome assembly, structural variation
○​ Microbiome research, haplotyping, splicing isoforms
Next-Generation Sequencing (NGS) Data Analysis Outlook for NGS and TGS
Raw Data Conversion
●​ TGS and NGS revolutionized genome sequencing, enabling:
●​ Starts with fluorescence/luminescence images from the flow cell ○​ De novo sequencing
●​ Image → sequence via base calling algorithms ○​ Whole-genome resequencing
○​ Assigns read sequences and quality scores ○​ Genetic diversity studies
●​ Advanced software can improve: ●​ Clinical potential: Diagnostics, mutation detection, personalized
○​ Ambiguity handling medicine
○​ Low-quality base removal ●​ Future developments:
○​ Training-based improvements ○​ Enhanced data processing tools
○​ Better accuracy, cost-efficiency, and speed
Alignment & Assembly ○​ Broader applications in evolution, epidemiology, and
●​ Short reads make alignment difficult disease research
●​ Challenges:
○​ Repetitive sequences
○​ Pseudogenes Summary Table
○​ Gene family homologies Technology Key Feature(s) Read PCR Applications
Length Required
Accuracy Considerations:
●​ NGS reads have higher individual error rates
●​ Coverage depth (20–60×) needed for accuracy:
○​ E.g., 20–30× for Illumina resequencing Helicos Virtual terminator, ~35 nt No Expression, ChIP, copy
direct RNA seq number, degraded DNA
●​ Consensus sequences increase overall accuracy
●​ Coverage gaps and ambiguous alignments reduce confidence
PacBio SMRTbell, movie ~1300 No Assembly, methylation,
capture, epigenetic bp microbiology
NGS Software Tools info

Life Tech Quantum dot + Varies No Assembly, haplotyping,

Alignment & Assembly: FRET metagenomics

●​ Reference alignment: Zoom, MAQ, Mosaik, SOAP, SHRiMP

●​ De novo assembly: Edina, EULER-SR, SHARCGS, SSAKE, Nanopore ssDNA through αHL >5 kbp No Long-read sequencing,
Velvet, SOAPdenovo pore, ionic current fast sample prep
readout
●​ Commercial Suites: DNAStar, SoftGenetics, CLC bio
○​ Allow alignment visualization, genome annotation,
variant analysis
 Characterization of Nucleic Acids and Restriction Endonucleases (Type II)
Proteins A. Historical Background

NUCLEASES ●​ Discovered from the observation that bacteria resist phage

infection.
Nucleases are enzymes that degrade DNA or RNA by hydrolyzing
●​ These enzymes degrade foreign phage DNA, but protect their
phosphodiester bonds. They are classified into:
own DNA via methylation.

1. Endonucleases B. Types of Restriction Endonucleases

●​ Cut internal bonds within a polynucleotide chain.
●​ Can attack from the 5’ or 3’ end of the linkage. Type Cleavage Location Biotech Use
●​ May be specific to single- or double-stranded DNA, or both.
Type I Cleaves far from recognition site (up to Limited
1000 bp)
2. Exonucleases
●​ Remove nucleotides one at a time from either the 5’→3’ or 3’→5’
Type III Cleaves close to recognition site (20–30 Limited
end of DNA. bp)

3. Specificities of Nucleases Type II Cleaves at the recognition site Widely used in molecular
●​ DNases: Degrade DNA. biology
●​ RNases: Degrade RNA.
●​ RNase H: Degrades the RNA strand of a DNA-RNA hybrid.
●​ May generate either 5' nucleotides or 3' nucleotides, depending ●​ Type II enzymes are the standard for laboratory use and recognize
on which ester bond is cleaved. palindromic sequences (4–8 bp long).
●​ Many target hexanucleotide sequences (6 bp).
4. Examples of Nucleases ●​ Some recognize degenerate sequences, e.g., HinfI recognizes
GANTC, allowing variation in one position.​
●​ Bal31: Removes nucleotides from both strands of double-stranded
DNA.
●​ Exonuclease III: Degrades one strand of a double-stranded DNA.
●​ S1 Endonuclease: Cleaves only single-stranded nucleic acids.
●​ DNase I: Nonspecific; cleaves both single- and double-stranded
DNA into mononucleotides or short oligonucleotides.
●​ Restriction Endonucleases: Recognize specific sequences and
cleave DNA at internal positions.
●​ Ribonuclease: Rapidly degrades RNA into ribonucleotide
subunits.
Variants of Restriction Endonucleases Analysis of Restriction Digest Products
1. Isoschizomers
●​ Recognize and cut at the same site. 1. Restriction Digest
●​ Example: BspEI and AccIII. ●​ Performed in a microcentrifuge tube with:
2. Neoschizomers ○​ Template DNA
●​ Recognize the same sequence, but cut at different positions. ○​ Specific restriction enzyme
○​ Mg²⁺ (as cofactor)
●​ Example: NarI and SfoI.
●​ Produces fragments depending on cut sites.
3. Isocaudomers
●​ Recognize different sequences, but produce the same sticky
ends. 2. Gel Electrophoresis
●​ Example: NcoI and PagI. ●​ Separates fragments based on size.
4. Methylation-Dependent Restriction Enzymes ●​ Smaller fragments travel faster through the gel.
●​ Some only cleave when the recognition site is methylated.​
3. Restriction Map
●​ Linear diagram of DNA showing locations of restriction enzyme cut
Restriction-Modification (R-M) System sites.
●​ Helps identify DNA regions.​
●​ A host defense mechanism in bacteria. Often followed by Southern blotting for specific fragment
●​ Paired enzymes: detection.
○​ Restriction Endonuclease: Cuts foreign DNA.​
Methylase: Methylates host DNA at the recognition site, Summary Key Points
preventing cleavage.
●​ Hemimethylation during replication protects DNA temporarily ●​ Nucleases: Cut DNA/RNA internally or from the ends.
until full methylation occurs. ●​ Type II Restriction Enzymes: Crucial in DNA manipulation, cut at
specific palindromic sites.
Frequency of Recognition Sites in DNA ●​ Isoschizomers, Neoschizomers, Isocaudomers: Variants with
distinct recognition or cutting behaviors.
●​ Based on probability: ●​ R-M System: Protects bacterial DNA through methylation.
○​ Tetranucleotide (4 bp): Every 256 bp (4⁴). ●​ Frequency & Mapping: Recognition sites appear at predictable
○​ Hexanucleotide (6 bp): Every 4096 bp (4⁶).​ intervals, enabling fragment analysis and mapping.
●​ Example (λ DNA, 49 kb):
○​ Expected ~12 hexanucleotide sites.
○​ Actual: 6 sites for BglII, 5 for BamHI, 2 for SalI.​

●​ Conclusion: Restriction sites are not evenly distributed.

 Enzymes in Nucleic Acid Manipulation & 4. Reverse Transcriptase
Hybridization Techniques
●​ Converts RNA to complementary DNA (cDNA).
●​ Used in RT-PCR to assess RNA levels and gene expression
DNA Polymerases profiling.
DNA polymerases are enzymes that synthesize a new DNA strand ●​ Vital for studying RNA viruses and transcriptional activity.​
complementary to an existing DNA or RNA template. Most require a
double-stranded primer to initiate polymerization.
DNA Modifying Enzymes & Ligases
1. DNA Polymerase I (E. coli)
●​ Functions: A. DNA Modifying Enzymes
○​ Polymerase activity: Synthesizes DNA on nicked or
single-stranded regions. Enzyme Function
○​ 3′→5′ Exonuclease: Proofreading function.
○​ 5′→3′ Exonuclease: Removes and replaces DNA strands
ahead of the polymerase.
Alkaline Removes 5′ phosphate groups from DNA (from E. coli, calf
●​ Applications: DNA repair, synthesis, and replacing damaged
Phosphatase intestine, shrimp).
strands.
●​ Dual activity: Both synthesis and degradation of DNA.
Polynucleotide Adds 5′ phosphate groups (from T4-phage-infected E.
Kinase coli).
2. Klenow Fragment
●​ Derived from mild proteolysis of DNA Polymerase I. Terminal Transferase Adds deoxynucleotides to 3′ ends of DNA (from calf
●​ Large fragment: Retains polymerase + 3′→5′ exonuclease thymus).
activity.
●​ Small fragment: Has 5′→3′ exonuclease activity.
●​ Application: DNA end-filling, DNA sequencing.
B. DNA Ligase
●​ Limitation: Cannot extend beyond the filled-in nick (no 5′→3′
exonuclease). ●​ Natural role: Repairs single-stranded DNA breaks during
replication or repair.
●​ Lab application: Joins two DNA fragments or circularizes DNA.
3. Taq DNA Polymerase ●​ Mechanism: Forms phosphodiester bonds to seal DNA ends.
●​ Isolated from Thermus aquaticus. ●​ Types of ligation:
●​ Thermostable: Remains active during high-temperature ○​ Joining two fragments.
denaturation (94°C). ○​ Closing nicks in one strand.
●​ Essential for: Polymerase Chain Reaction (PCR).
Nucleic Acid Hybridization B. Northern Hybridization (Northern Blotting)
Hybridization is the base-pairing of complementary nucleic acid strands, ●​ Target: RNA
either DNA:DNA or DNA:RNA. When one strand is labeled, it acts as a
probe to detect complementary sequences in unknown samples. ●​ Purpose: Detect RNA transcripts (gene expression)
●​ Probe: Labeled DNA (complementary to RNA)
●​ Precautions:
A. Southern Hybridization (Southern Blotting) ○​ RNA is unstable; must avoid RNase contamination.
●​ Target: DNA ○​ DEPC treatment for reagents/equipment.
●​ Steps: ●​ Denaturation:
○​ Genomic DNA extraction ○​ Before electrophoresis: Heat at 55°C with formamide +
○​ Restriction enzyme digestion formaldehyde.
○​ Separation via agarose gel electrophoresis ○​ During electrophoresis: Include formaldehyde in gel.
○​ Denaturation & depurination (HCl + NaOH) to fragment ●​ Electrophoresis tip:
DNA ○​ Avoid ethidium bromide → reduces signal.
○​ Transfer to membrane (nylon) ●​ Transfer & Fixation:
■​ Ascending capillary transfer ○​ Capillary or vacuum blotting onto nylon membrane.
■​ Descending vacuum transfer ○​ Fix by UV or baking at 80°C.
○​ Probe hybridization ●​ Application:
○​ Detection​ ○​ Assess gene transcription levels
○​ Study expression patterns across conditions
●​ Key Parameters:
○​ Agarose gel %:
■​ 0.7–1.2% optimal for 0.2–20 kbp Summary Table of Blotting Techniques
■​ <0.7%: fragile gels Feature Southern Blot Northern Blot
■​ 1.2%: poor transfer efficiency
○​ Voltage/time: Target DNA RNA
■​ High voltage = faster, less resolution
■​ Low voltage + longer run = better resolution
Probe Labeled DNA Labeled DNA
●​ Fixation:
○​ Baking at 80°C for 2h
○​ UV irradiation Sample treatment Restriction digestion Denaturation (heat +
chemicals)
○​ Ensures long-term storage.
●​ Applications: Electrophoresis Agarose Denaturing agarose
○​ Detect gene variants gel
○​ Restriction Fragment Length Polymorphism (RFLP)
analysis Fixation UV or baking at 80°C UV or baking at 80°C
○​ Genetic mapping
Applications Gene mapping, RFLPs, Gene expression
mutations analysis
 RFLP, AFLP, and Probe Detection Amplified Fragment Length Polymorphism (AFLP)
Techniques ●​ AFLP is a high-resolution DNA fingerprinting technique
combining RFLP specificity and PCR sensitivity
●​ Can analyze hundreds to thousands of genomic loci
Restriction Fragment Length Polymorphism (RFLP) simultaneously
Analysis ●​ Does not require prior knowledge of genome sequences →
ideal for nonmodel species
●​ RFLP is a variation in homologous DNA sequences that results in
fragments of different lengths after digestion with specific AFLP Workflow
restriction endonucleases.
●​ Caused by Variable Number Tandem Repeats (VNTRs).
Step 1: Restriction Digestion
●​ Each individual's RFLP pattern is unique → used in DNA
●​ DNA cut with two enzymes: one rare-cutter (e.g., EcoRI) and one
fingerprinting, paternity testing, and disease gene mapping. frequent-cutter (e.g., MseI)
RFLP Workflow Step 2: Adapter Ligation
1.​ Genomic DNA extraction ●​ DNA fragments ligated to double-stranded oligonucleotide
adapters with cohesive ends complementary to restriction sites
2.​ Digestion with restriction enzymes
3.​ Gel electrophoresis to separate DNA fragments Step 3: Pre-selective PCR
4.​ Southern blotting ●​ Amplifies heterosite fragments with adapter sites for both
5.​ Hybridization with a labeled probe enzymes

Application Step 4: Selective PCR

●​ Detects genetic polymorphisms
●​ Can serve as a genetic marker if close to a disease gene ●​ Uses primers with 1–3 extra nucleotides at 3' end → reduces
complexity

Enhancement: CAPS Assay ●​ Allows visualization via:

○​ Polyacrylamide Gel Electrophoresis (PAGE) with
●​ Cleaved Amplified Polymorphic Sequence (CAPS): Uses PCR fluorescent/radioactive labeling
amplification before restriction enzyme digestion ○​ Capillary Electrophoresis (CE) with fluorescent primers
●​ Faster and requires less starting DNA​
Advantages of AFLP
●​ Highly reproducible, robust, and sensitive
●​ High-resolution genotyping
●​ Cost- and time-efficient
●​ Used in: systematics, population genetics, pathotyping, QTL
mapping
Detection Methods: Probes & Labeling 2. Nonradioactive Labeling
Principle of Hybridization a. Biotin-Avidin System
●​ Single-stranded probe DNA or RNA binds to complementary
●​ Incorporates biotin-labeled dUTP
sequences on target DNA/RNA
●​ Stability depends on degree of complementarity and hybridization
●​ Detected by avidin-fluorophore conjugates
conditions (e.g., temperature)
RFLP Probe b. Chemiluminescent Detection
●​ A labeled DNA sequence that binds to digested DNA fragments ●​ Probes complexed with horseradish peroxidase (HRP)
separated via gel electrophoresis ●​ HRP degrades luminol, emitting light detectable on film
●​ Reveals blotting pattern unique to genotype at a specific locus
●​ Usually made from short, single- or low-copy genomic DNA or cDNA Advantages:
clones
Uses ●​ Safe
●​ Genome mapping ●​ Environmentally friendly
●​ Genotyping ●​ Comparable sensitivity to radioactive methods
●​ Forensics
●​ Paternity testing
●​ Hereditary disease diagnostics

Labeling Methods for Probes

1. ☢️ Radioactive Labeling (32P)
Methods:
●​ Nick Translation:
○​ Uses DNA polymerase I to incorporate 32P-labeled
nucleotides
○​ May cause DNA cleavage
●​ End Filling:
○​ Uses Klenow fragment to fill sticky ends with labeled
nucleotides
○​ Gentler than nick translation
●​ Random Priming:
○​ Uses hexameric primers and Klenow fragment
○​ High sensitivity, useful for short probes
Detection:
●​ Autoradiography with X-ray film
●​ May use an intensifying screen to enhance signal
 Summary Table ARRAY-BASED HYBRIDIZATION
Technique DNA
Requirement
Speed Sensitivity Speci
ficity
Applications
🔷 Dot Blot and Slot Blot Hybridization
●​ Principle: Samples are immobilized on nitrocellulose/nylon
RFLP High Slow Moderate High DNA membrane in a geometric array.
fingerprinting,
paternity, gene ○​ Dot blot: Regular shape using suction manifold.
mapping ○​ Slot blot: Rectangular shape, also suction-assisted.
●​ Sample Type:
CAPS Low Fast High High Polymorphism ○​ Pure samples = higher specificity.
(PCR-based) detection ○​ Unpurified samples = may have background signals.
●​ Interpretation: Straightforward; presence of signal indicates
hybridization (yes/no result).
AFLP Moderate Fast Very High High Evolution,
population ●​ Limitation: No size information of hybridized fragments.

🔷 Macroarrays
genetics, QTL
mapping

Probe Varies Modera Very High High Blotting, ●​ Definition: Reverse dot/slot blot on a larger scale.
Detection te genotyping, ●​ Features:
diagnostics ○​ Visible without magnification.
○​ Contains thousands of target probes.
Radioactive Varies Slow Very High High Autoradiography
Labeling ○​ Printed or dot-blotted and dried for storage.
○​ Lower density than microarrays.
●​ Detection: Uses radioactive or chemiluminescent signals.

🔷 Microarrays
Nonradioac Varies Fast High High Safer hybridization
tive detection
Labeling

●​ Definition: Miniaturized, high-density dot/slot blots.

●​ Format: Gridlike pattern with hundreds to thousands of probes on
a solid surface (e.g., microscope slide).
●​ Applications:
○​ Transcript profiling: Comparing mRNA expression
between samples.
●​ Hybridization Workflow:
○​ Labeled cDNAs (from mRNA) are hybridized to arrays.
○​ Microarrays: Dual-color labeling (control vs. experiment).
○​ GeneChips: Single-color labeling per chip.
●​ Output: Fluorescence intensity → gene expression ratio.
Microarray-Manufacturing Technologies Sample Processing & Detection (Microarrays)
Sample Preparation
1. Synthesis Method (In Situ Synthesis) ●​ Isolate mRNA rapidly to preserve expression profile.
●​ Techniques: e.g., Laser Capture Microdissection (for isolating
●​ Uses photolithography and DNA chemistry. specific cells).
●​ Key example: Affymetrix GeneChips (400,000 probes in 1.6
cm²). Hybridization Conditions
●​ Advantages:​ ●​ Requires sensitive and specific probe-target binding.
●​ Conditions depend on melting temperature (Tm):
○​ Direct from sequence database. ○​ 4–5× SSC buffer: 60–65°C
○​ High precision and low variation. ○​ 50% formamide: 42°C (formamide lowers Tm)
●​ Disadvantages: ○​ Fluorophores reduce Tm → less stringent conditions
○​ Requires expensive photomasks. ●​ Additives to reduce nonspecific binding:​
Salmon sperm DNA, poly[dA], tRNA, SDS
2. Delivery Method (Microspotting)
Detection Methods
●​ Deposits pre-made cDNAs onto arrays. ●​ Fluorescence (laser-induced, read via confocal optics)
●​ Uses pins, capillaries, or tweezers for spotting. ○​ Resolution: 11.25 μm, scanning time: 15 mins for 1-cm²
●​ Up to 10,000 cDNAs in 3.6 cm².​ chip.
●​ Surface Plasma Resonance (for unlabeled probes).
●​ Advantages: ●​ Real-Time Monitoring:
○​ Cost-effective and easy prototyping.​ ○​ CCD camera imaging.
○​ Evanescent wave excitation (detects only surface-bound
●​ Disadvantages:​ fluorophores).

○​ Needs sample prep (amplification, purification, storage). WESTERN BLOT

●​ Proteins are separated by electrophoresis and blotted onto a
3. Drop-on-Demand (Ink Jetting) membrane for detection.
●​ Allows protein identification using ligands like antibodies.
●​ Uses piezoelectric nozzles to deliver reagents.
●​ Non-contact, high-throughput gridding. Steps
●​ Can deposit cDNAs, genomic DNA, antibodies, etc. 1.​ Protein separation (e.g., SDS-PAGE or IEF)
●​ Useful for real-time gene expression applications. 2.​ Transfer to membrane (e.g., nitrocellulose)
3.​ Probe with antibody
4.​ Detect signal (e.g., enzyme-labeled secondary antibodies)
Detection Probes Summary Chart
●​ Primary antibody → binds to target protein.
●​ Secondary antibody → enzyme-labeled (e.g., protein A, G, Technique Target Sample Type Detection Info Gained
streptavidin-biotin). Molecule
●​ Requires nondenaturing conditions if native protein function is
essential. Dot/Slot Blot Nucleic Pure/Unpurified Signal at spot Yes/No
Acids hybridization
Applications
●​ Immunoblotting: Detects specific proteins. Macroarray Nucleic Nucleic acid mix Radioluminescent Hybridization,
●​ Clinical diagnostics: Common in HIV/AIDS testing. Acids low density
●​ Research: Analyze protein modifications (e.g., phosphorylation),
perform sequencing, or use proteins as immunogens. Microarray Nucleic mRNA-derived Fluorescence Gene
Acids cDNA expression
profiling
Gel Electrophoresis for Protein Separation
Western Blot Proteins Protein extracts Antibody/Enzyme Protein ID,
tag modifications
1. SDS-PAGE
SDS-PAGE Proteins Denatured Coomassie/silver Size-based
●​ Proteins denatured with SDS and β-mercaptoethanol.
separation
●​ Separation based on size only.

Native PAGE Native Native (buffered) Depends on dye Size/charge or

2. Native PAGE / IEF Proteins pI separation

●​ Proteins retain native conformation.

●​ Separation based on size and charge.
●​ Maintains protein-protein or protein-DNA interactions.

3. Isoelectric Focusing (IEF)

●​ Uses pH gradient gel.
●​ Proteins migrate until reaching their isoelectric point (pI).
●​ Best for hydrophilic proteins; hydrophobic proteins need
detergents.
Methods for Transferring Proteins into Membranes Types of Membranes for Blotting
A. Passive Transfer (Diffusion-Based) Membrane Type Properties
●​ Involves placing membrane sheets on one or both surfaces of
the gel. Nitrocellulose Most common; high protein binding, low
●​ Proteins diffuse from gel to membrane due to high membrane background, fragile.
affinity.
●​ Works best with agarose gels (larger pores), less efficient with Nylon Stronger, high capacity; charged nylon
polyacrylamide gels. (charged/uncharged) enhances binding further.
●​ Enhancement methods:
○​ Capillary action (filter paper stack)
○​ Vacuum-assisted flow PVDF (polyvinylidene Excellent strength, high protein binding, must
difluoride) be wetted with methanol before use.
Limitation: Inefficient for proteins separated by IEF (proteins
at isoelectric point = no net charge). ⚠️ PVDF must remain wet during blotting and detection.
Protein Visualization and Staining
B. Electroblotting (Electrotransfer) ●​ Use pre-stained molecular weight markers for tracking transfer.
●​ Proteins driven from gel to membrane by an electric field. ●​ Suitable stains for nitrocellulose and PVDF:​
●​ Setup:
○​ Gel-membrane sandwich immersed in buffer tank with ○​ Amino black, India ink: good sensitivity.
electrodes, or ○​ Ponceau: least sensitive but reversible (0.1 N NaOH).​
○​ Semidry blotting: filter paper stacks (wet with different
buffers) + electrodes. ●​ PVDF bands turn translucent in methanol – viewable with
●​ Advantages:
○​ Faster and sharper than passive methods. ●​ ⚠️
lightbox.
Nylon membranes are not suitable for total protein stains.
○​ No cooling system needed in semidry blotting.
●​ Risks:
○​ "Blow through": Proteins pass through membrane.
Blocking and Detection in Western Blotting
■​ Influenced by: membrane pore size, field strength, A. Blocking Unused Binding Sites
protein size. ●​ Prevents non-specific binding during antibody detection.
○​ Solution: Use a lower electric field, allow time for ●​ Blocking agents:
protein binding.​ ○​ Ovalbumin/gelatin: best results, lowest background.
○​ Fat-free milk powder: economical and effective.
○​ Tween-20: simple, but may increase background or
detach proteins.
B. Immunodetection of Proteins 3. Other Ligand-Based Detections
●​ Lectins for glycoproteins.
1. Indirect Method ●​ DNA/RNA probes for nucleic acid-binding proteins (renatured
after blotting).
●​ Use of: ●​ Protein-protein interaction studies – immunologic or radioactive
○​ Primary antibody (Ab) – polyclonal or monoclonal. detection.
○​ Secondary antibody – enzyme-labeled (commonly HRP
or AP). Applications of Western Blotting
○​ Alternative: Protein A/G for broader IgG recognition. ●​ Used in protein chemistry, enzymology, oncology.
●​ Detects:
Polyclonal Abs recognize multiple epitopes = better binding.​ ○​ Isoenzymes
Monoclonal Abs may struggle with SDS-denatured proteins if ○​ Tumor markers
epitope is conformational. ○​ Protein localization and expression profiles

2. Labeling Techniques
Enzyme-Linked Immunosorbent Assay (ELISA)
●​ Enzyme Labels: HRP, AP (commonly used)
●​ Chromogens: produce colored precipitates for detection. A. Principle
●​ Bridge System: adds antienzyme Ab to increase sensitivity. ●​ Based on antigen-antibody specificity.
●​ Biotin-Avidin System: multiple enzyme molecules per Ab. ●​ Antigen immobilized on solid support (e.g., polystyrene microtiter
plate).
C. Enhanced Detection Techniques ●​ Detection: enzyme-labeled antibody +
chromogenic/fluorogenic/chemiluminescent substrate.
1. Chemiluminescence (CL)
B. Common Enzymes and Reactions
●​ Enzyme-activated light-emitting substrate (e.g., luminol for HRP).
●​ Detection via photographic film + densitometry for Enzyme Substrate Detection
quantification.
●​ Drawback: Exposure time needs optimization. ALP p-nitrophenyl phosphate Yellow (405 nm)

2. Radioactivity HRP Chromogens or luminol Color or light emission

●​ Radioactively labeled proteins or ligands (e.g., ¹²⁵I-Ab). β-galactosidase Fluorogenic substrates Fluorescence
●​ Detected by X-ray film.
●​ Drawback: Handling and disposal of radioactive materials.
●​ Chemiluminescence: Greater sensitivity.
●​ Reaction time: 30–60 mins, stopped with acid/base.
C. Advantages of ELISA
Genome and Transcriptome Analysis
1.​ Simple and rapid.
2.​ High specificity and sensitivity. ●​ Next-Generation Sequencing (NGS) has greatly improved the
3.​ High-throughput and eco-friendly. ability to diagnose and predict diseases.
4.​ Cost-effective (low-cost reagents). ●​ Predicting disease phenotypes is challenging due to:
○​ Multiple genes causing the same phenotype.
D. Limitations ○​ One gene being linked to various phenotypes.
●​ Labor-intensive to prepare Abs. ○​ Variable effects of mutations depending on gene-gene,
●​ Requires expensive media for antibody production. gene-protein, and environmental interactions.
●​ False positives/negatives due to:
Interactome
○​ Poor blocking ●​ The complete set of physical interactions between molecules
○​ Antibody instability (genes, proteins, metabolites).
●​ Translates phenotypic effects of genotypes + environmental
factors.

What is GWSS?
Genome-Wide Sampling Sequencing (GWSS) = cost-effective strategy
for detecting genetic polymorphisms, especially SNPs (single
nucleotide polymorphisms).

Why GWSS?
●​ ~150 million SNPs identified in the human genome.
●​ Whole-genome resequencing is expensive and unnecessary
for all SNP genotyping.

GWSS Techniques Include:

1.​ Reduced genome complexity sequencing
2.​ Reduced genome representation sequencing
3.​ Selective genome target sequencing

Common Steps in GWSS:

1.​ DNA digestion with restriction enzymes
2.​ Ligation with adapters
3.​ PCR amplification for sequencing

Sequencing Platforms:
●​ Illumina
●​ Ion Torrent
Categories of GWSS Methods GGRS Specifics:
Category Description ●​ Simpler, rapid, reproducible
●​ Uses identical barcode adapters on both ends
GWSS without size selection e.g., GBS, GGRS ●​ Reduces cost with gel electrophoresis size selection
●​ Combines adapter removal & PCR cleanup into one step
GWSS with semi-size selection e.g., RAD-seq, DDRAD-seq

GWSS with size selection e.g., RRS, RRLs

GWSS With Semi-Size Selection
Key Methods:
GWSS with selective amplification e.g., CRoPSTM ●​ RAD-seq (Restriction Site Associated DNA)
●​ Paired-end RPLs
GWSS Without Size Selection ●​ DDRAD-seq
●​ Flexible & scalable GBS
●​ GBS (Genotyping-by-Sequencing)
●​ GGRS (Genome Reducing and Sequencing) RAD-Seq Highlights:
●​ Detects SNPs near restriction sites
Procedure: ●​ Useful for: population genetics, association mapping, allele
●​ Digest DNA with restriction enzymes frequency estimates
●​ Ligate barcoded adapters ●​ Sequences transcribed & non-transcribed loci
●​ PCR amplification of fragments
Procedure:
Adapter Design in GBS: ●​ Adapter ligation → random shearing
●​ Uses a barcode adapter + common adapter ●​ Two rounds of size selection
●​ Barcode = 4–8 bp on 3’ end ●​ End repair, dA tailing, and Y adapter ligation
●​ Sticky ends generated by ApeKI ●​ Paired-end sequencing improves SNP detection
●​ Designed to avoid regenerating restriction sites
GWSS With Size Selection
Two-Enzyme GBS:
Key Methods:
●​ Uses rare and common cutters (e.g., PstI & MspI)
●​ Forward adapter = barcoded (PstI) ●​ RRS (Reduced Representation Shotgun Sequencing)
●​ Reverse adapter = Y-type (MspI) ●​ RRLs (Reduced Representation Libraries)
●​ Only PstI-MspI fragments are amplified ●​ GBS with enzyme digestion
●​ Type IIB RAD​
Procedure: Key Takeaways:
●​ Restriction enzyme digestion
●​ Size selection using polyacrylamide gel ●​ GWSS is a powerful, cost-effective alternative to full-genome
●​ Collect DNA fragments (e.g., 100–200 bp) sequencing.
●​ Adapter ligation → PCR → Sequencing ●​ It enables high-throughput SNP detection for both known and
unknown genomes.
●​ Proper library design, enzyme selection, and size selection
GWSS With Selective Amplification methods are crucial to success.
Key Methods: ●​ Each GWSS method offers trade-offs in complexity, cost, and
●​ CRoPSTM (based on AFLP) accuracy—choose based on your study goal.
●​ Scalable GBS
●​ Whole Exome Sequencing (WES)
Genome-Wide Association Studies (GWAS) and
CRoPSTM Highlights: Transcriptome Sequencing
●​ Digestion → adapter ligation → PCR (pre + selective)
●​ Separated on gel or sequenced GENOME-WIDE ASSOCIATION STUDIES (GWAS)
Identification of SNPs
WES Highlights: ●​ GWAS analyze associations between common genetic variants
●​ Targets 2%–3% of genome (covers >98% of exons) (especially SNPs) and phenotypic traits.
●​ Construct WGS library → gel electrophoresis ●​ They identify SNPs statistically associated with complex diseases
●​ Hybridize with exome arrays by comparing the frequency of allelic variants in case vs. control
●​ Recover and sequence exon-rich fragments samples.
●​ Confounders like ethnicity and relatedness are controlled.
Challenges in GWSS ●​ A significant difference in SNP frequency suggests a
1.​ Inconsistent reads per sample risk/protective allele.
2.​ Uneven marker distribution
3.​ Missing data GWAS Achievements
4.​ Low genotype accuracy ●​ Enabled by advances in genotyping tech and statistical methods.
Solutions: ●​ Over the past 20 years, GWAS identified risk factors for numerous
●​ Use one-sample-one-library approach complex diseases.
●​ Optimize primer efficiency ●​ As of March 2019, NHGRI GWAS Catalog lists 126,603 SNP-trait
●​ Prefer bead-based size selection over gels associations from 3841 studies.
●​ Choose **enzymes with: ●​ Also contributed to pharmacogenomics—linking SNPs with drug
○​ Rarely mutated sites efficacy and toxicity.​
○​ Resistance to methylation (Dam, Dcm, CpG)
●​ Ensure even fragment size distribution
●​ Avoid artifacts via better ligation control
Interpreting GWAS Variants Omics Integration
●​ Due to linkage disequilibrium (LD), most GWAS hits are not
causal. ●​ Bridging the genotype-phenotype gap requires combining:
●​ Only ~5% of lead SNPs are likely causal; average distance from ○​ DNA → RNA → Protein → Metabolite → Flux
causal variant is 14 kb. ●​ Multi-omics data integration is essential for advancing clinical
●​ Post-GWAS steps: applications.
○​ Dense genotyping
○​ Resequencing
○​ Imputation TRANSCRIPTOME SEQUENCING
○​ Comprehensive variant mapping
●​ Transcriptome = full set of RNA transcripts (coding & noncoding).
EPIGENETIC INTEGRATION IN GWAS ●​ Provides real-time gene expression data.
●​ Applications:
What is Epigenetics? ○​ Cell cycle, stem cell biology, tissue regeneration
●​ Epigenetics: chemical modifications (epigenome) that regulate ○​ Disease diagnostics and drug discovery
gene expression without altering DNA sequence. ○​ Host-pathogen interaction
●​ Key modifications:
○​ DNA methylation
○​ Histone modification Whole Transcriptome Sequencing Methods
○​ Chromatin remodeling
1. Whole Transcriptome Shotgun Sequencing (WTSS)
Environmental Influence and Phenotypic Plasticity Four main approaches:
●​ Epigenome reflects environmental exposure (e.g., nutrition, ●​ WTSS
oxidative stress). ●​ Tag/target sequencing with restriction digestion
●​ This leads to phenotypic plasticity, explaining variable disease ●​ Tag/target sequencing without restriction digestion
susceptibility. ●​ Other developments
●​ Abnormal plasticity is linked to cancer, neurodegenerative, and
autoimmune disorders. 2. Source Material & Library Prep
●​ Total RNA, polyA+ or polyA− RNAs used.
GWAS + Epigenetic Annotation ●​ Adapters added via cDNA synthesis or ligation.
●​ Integrating SNP data with epigenetic maps improves ●​ Amplification done using cloning or PCR.
understanding of how genetic variation leads to disease.
●​ Helps build a link from DNA sequence → epigenome →
phenotype.
●​ Mapping human epigenome and correlating environmental
exposures is complex and needs collaborative studies.
EST (EXPRESSED SEQUENCE TAG) SEQUENCING EST vs RNA-SEQ
EST Library Construction Feature EST RNA-Seq
●​ Short, single-pass cDNA reads from random clones.
●​ Can study: Sequencing Method Sanger NGS (e.g., Illumina)
○​ Exclusively expressed genes
○​ Co-expression patterns Throughput Low High
○​ Housekeeping genes
○​ Differentially expressed genes Cloning Requirement Required Not required

EST Sequencing Workflow (Fig. 12.9A–C) Data Output Limited Extensive

●​ Uses Sanger sequencing.
●​ Oligo(dT) primers for first-strand synthesis; switching primer Cost Higher Lower
enables full-length capture.
●​ Amplification → restriction digestion → cloning into E. coli. Popularity Declining Dominant method

a. Full-Length cDNA Library (Fig. 12.9B)

●​ Five-step cloning using pUC19, oligo(dC), HindIII, and oligo(dG)
for second-strand synthesis. RNA-SEQ DATA ANALYSIS

b. 3′-Directed cDNA Library (Fig. 12.9C) Workflow

●​ Uses only pUC19 vector primers. 1.​ Raw data → short reads
●​ Captures 3′ mRNA ends—ideal for gene assignment and profiling. 2.​ Read alignment to genome or transcriptome
3.​ Read counting → expression profile
4.​ Normalization
RNA-SEQ PROCEDURE (NGS-BASED 5.​ Statistical analysis → differential gene expression
TRANSCRIPTOMICS)
●​ Uses Illumina Genome Analyzer I. Key Notes
●​ Primer types: oligo(dT) and template-switching. ●​ For eukaryotes, align to reference transcriptome (if available) or
●​ Produces full-length cDNA with PCR-ready sequences. genome.
●​ Fragmented (100–300 bp), ligated with adapters, PCR amplified ●​ Spliced-read mapping tools help resolve exon-intron structures.
for sequencing. ●​ Normalization adjusts for:
Applications ○​ Gene length
●​ Detects transcript levels, isoforms, and polymorphisms. ○​ Sequencing depth
●​ Enables genome-wide polyA site profiling and alternative
polyadenylation analysis.​
RNA-SEQ CHALLENGES MPSS (Massively Parallel Signature Sequencing)
Technical Challenges
●​ High-throughput, gel-free method.
●​ Fragmentation bias: Ends of transcripts may be lost. ●​ Uses DpnII and BbvI to create 17 bp tags cloned on microbeads.
●​ Length bias: Longer RNAs yield more reads. ●​ Enables detection of low-abundance transcripts.
●​ Transcriptome composition bias: Highly expressed genes ●​ Inspired development of NGS platforms: Roche/454 FLX,
dominate read counts. Illumina/Solexa, ABI SOLiD, Helicos, PacBio.
Solutions
●​ Bioinformatics tools adjust read counts and normalize data.
●​ Corrected RNA-seq data shows strong correlation with qRT-PCR
NGS Integration
and microarrays. ●​ DGETP: Adapts SAGE to Illumina/Solexa without di-tags.
Computational Demands ●​ Tags flanked by Illumina GEX Adapters.
●​ RNA-seq requires 10–20× more reads than tag/target methods. ●​ High-throughput SuperSAGE: With Illumina or SOLiD platforms.
●​ Large data sets pose bioinformatics and computational
challenges. Tag/Target Elongation Techniques
●​ 3' End cDNA Amplification: Uses anchored oligo(dT) primers,
WHOLE TRANSCRIPTOME TAG/TARGET restriction digestion, Y-shaped adapter ligation.
●​ rSAGE: Uses 64 nt primers with 30 Ts, digested with NlaIII, 5'
SEQUENCING REVIEWER adapter ligation, PCR.
●​ PATs (PolyA Tags): Uses switching primers with restriction sites,
WITH RESTRICTION DIGESTION digested with NlaIII/TaiI, ligated with adapters.
○​ Sequences 100–600 bp long, improved gene assignment
SAGE (Serial Analysis of Gene Expression) accuracy.

●​ Provides a snapshot of the transcriptome. Problems with Restriction Digestion

●​ Uses magnetic beads with polyT tails to capture polyA+ RNA,
converted into cDNA. ●​ Enzymes may not cover all transcripts.
●​ cDNA digested with anchoring enzymes, ligated with adapters with ●​ PolyA junction cuts may yield uninformative tags.
tagging enzyme sites. ●​ PCR favors short fragments, creating length bias.
●​ Tags (~10-14 bp) are formed, joined into di-tags, concatamerized,
cloned, sequenced. Solutions
●​ Limitations: Short tags difficult to map to transcripts.
●​ Multiple rSAGE: Uses four enzymes—Tsp509I (AATT), NlaIII
Improvements (CATG), MspI (CCGG), DpnII (GATC).​
●​ LongSAGE: Uses MmeI for longer tags (21 bp).
●​ SuperSAGE: Uses EcoP15I for tags up to 26 bp. ○​ Covers 99.82% of X. tropicalis transcriptome.
●​ TALEST: Uses type IIs restriction enzyme site for fixed 16 bp ○​ Fragment sizes: 150–450 bp.
ESTs without PCR.
WITHOUT RESTRICTION DIGESTION NON-POLYADENYLATED RNA PROFILING
●​ Targets rRNA, small RNAs, histone mRNAs, bacterial mRNA.
Transcriptome Profiling of 3' Ends (PolyA+ RNA ●​ Add RNA adapter to 3' ends.
Enrichment) ●​ Remove 18S/28S rRNA with biotinylated probes.
●​ Remove small RNAs via size selection.
●​ Ligate 5' adapter post NlaIII digestion.
●​ 3PC (cDNA Circulation), 3' READS: ●​ Amplify and sequence using Roche 454.
○​ Fragment total/polyA+ RNA. ●​ Alternatively, deplete polyA+ RNA using oligo(dT) beads.
○​ Enrich fragments using oligo(dT) magnetic beads.
○​ Reverse transcription, adapter ligation, PCR. CIRCULAR RNA (circRNA) PROFILING
●​ CircRNAs are covalently closed, noncoding RNAs.
3'-End Profiling (PolyA+ cDNA Enrichment) ●​ Derived from self-splicing or spliceosome activity.
●​ RNase R degrades linear RNA, enriching circRNAs.
●​ 3' T-fill Method: ●​ Detected using custom algorithms.
○​ cDNA synthesized from fragmented RNA with biotinylated ●​ PolyA- enrichment reduces noise, increases detection.
oligo.
○​ RNase H treatment, second strand synthesis, polyA+
enrichment with Dynabeads. RNA METHYLATION PROFILING
○​ PCR and size selection.​
●​ RNA Bisulfite Sequencing:
●​ EXPRSS: ○​ Bisulfite converts cytosine to uracil.
○​ Uses oligo(dT) primers with Illumina P7. ○​ Sequencing reveals methylation sites.
○​ cDNA sheared by Covaris to ~200 bp, ligated to ●​ Aza-IP (Aza-Immunoprecipitation):
Y-adapters. ○​ Uses m5C-RMTs and 5-aza-C to form stable complexes.
○​ Avoids enzyme site limitation. ○​ Immunoprecipitate and sequence.
●​ miCLIP (Methylation Individual-Nucleotide-Resolution
3'-End Profiling With Custom Oligo(dT) Primers Crosslinking and IP):
○​ Uses NSUN2 methyltransferase.
●​ PAS-seq:
○​ Fragmented polyA+ RNA reverse transcribed with 5'-END PROFILING (G-CAP ENRICHMENT)
oligo(dT) and switching primers.
○​ Dual PCR rounds (3 + 15 cycles), size selection. ●​ CAGE (Cap Analysis of Gene Expression):
●​ PolyA-seq: ○​ DeepCAGE, PEAT, nanoCAGE, CAGEscan.
○​ Unfragmented polyA+ RNA reverse transcribed. ○​ BAP treatment to remove non-capped RNAs.
○​ RNase H treatment, second strand synthesis with N7 ○​ High-throughput identification of transcription start sites
primers. (TSSs).​
○​ 32 PCR cycles for sequencing.
 RNA Structurome & Metabolomics for GWAS High-Throughput Transcriptome-Wide Methods
●​ Combine RNase probing with Next-Gen Sequencing (NGS).
●​ Main methods using DMS for in vivo RNA structure analysis:
THE RNA STRUCTUROME 1.​ Structure-seq
2.​ DMS-seq
●​ RNA plays a key role in transcription, processing, translation, and 3.​ Mod-seq
regulation.
●​ It acts as a sensor for: Shared Sample Preparation Steps
○​ Proteins, RNAs, DNAs ●​ In vivo DMS modification
○​ Ligands ●​ Reverse transcription
○​ Temperature ●​ Ligation for PCR & NGS​
○​ Mutations
●​ RNA’s single-stranded (ss) nature allows formation of:
○​ Secondary structures: hairpins, three-way junctions Differences Among Methods
○​ Tertiary structures: pseudoknots, G-quadruplexes
●​ These structures regulate: Feature Structure-seq DMS-seq Mod-seq
○​ Gene expression
○​ Ligand sensing RT Priming Random Fragmented RNA Fragmented RNA
○​ Enzymatic activity hexamer (N6)

Probing RNA Structures Fragmentation None Random Random

hydrolysis hydrolysis
●​ Chemical probes:
○​ DMS (Dimethyl sulfate) RNA Adapter One side 3' ligation 3' + 5' ligation
○​ NAI (2-methylnicotinic acid imidazolide) Ligation
○​ 1M7 (1-methyl-7-nitroisatoic anhydride)​
5' Adapter Not needed No Biotin-oligo
Selection capture

Transcriptome-Wide RNA Structural Probing In Vivo Ligation Type Linear Circular Circular
Traditional Methods (intermolecular) (intramolecular) (intramolecular)

●​ Gel electrophoresis: low throughput (~150 nt).

●​ Capillary electrophoresis (CE): improved (~500 nt). Complexity Simple Moderate Complex

●​ Structure-seq: Less RNA processing → reduced degradation risk.

●​ All methods use Circligase for cDNA ligation but with known sequence
bias.
Output Analytical Techniques

●​ PCR amplifies cDNA for NGS. ●​ NMR spectroscopy

●​ Data used to analyze: ●​ Mass spectrometry
○​ Translation ●​ Generates high-resolution spectral data (>40,000 variables)
○​ Splicing ●​ Data analyzed using:
○​ Polyadenylation ○​ Univariate statistics
○​ miRNA regulation ○​ Multivariate statistics
○​ RNA stability & localization​ ●​ Goal: Identify biomarkers related to physiological or pathological
states.

METABOLOMICS FOR GENOME-WIDE Metabolomics-Integrated GWAS (mGWAS)

ASSOCIATION STUDIES (mGWAS) Basics of GWAS
●​ Traditional GWAS links genetic variants to disease endpoints.
●​ Limitations:
What is Metabolomics? ○​ Weak associations
●​ Study of all small molecules (<1500 Da) (metabolites) in a ○​ Poor predictive power for complex diseases
biological sample. ○​ Often fails to explain underlying mechanisms
●​ Metabolome: total set of small molecules in cells, tissues, or fluids.
●​ Provides intermediate phenotypes and uncovers disease Advantages of mGWAS
mechanisms beyond genomics. ●​ Uses metabolite levels (e.g., glucose, lipids, amino acids) as
traits.
Applications ●​ Improves identification of metabotype QTLs (mQTLs).
●​ Increases biological relevance of genotype-phenotype
●​ Tracks changes in metabolites from:
associations.
○​ Blood, urine, tissues, organs, cell cultures Process
●​ Reveals metabolic pathway dynamics in: 1.​ Profile metabolome using NMR/MS.
○​ Health and disease 2.​ Identify mQTLs.
○​ Personalized medicine 3.​ Link mQTLs to genomic variants via GWAS.

Takeaway: mGWAS bridges genotype and phenotype by incorporating

biochemical context, enabling:

●​ Accurate disease models

●​ Biomarker discovery
●​ Personalized healthcare strategies
Summary
Topic Key Takeaway

RNA RNA folding and structure govern function and gene

Structurom regulation. Modern methods (Structure-seq, DMS-seq,
e Mod-seq) allow high-throughput in vivo structure probing.

Metabolom Measures all small molecules to study metabolic

ics networks. Used to understand disease mechanisms and
complement genomics.

mGWAS Integrates metabolomics with GWAS to reveal functional

insights into genetic variants and disease traits.