MOLECULAR BIOLOGY TECHNIQUES AND APPLICATIONS

MLS 5321 MOLECULAR BIOLOGY II TECHNIQUES
AND APPLICATIONS
Bioinformatics is an interdisciplinary field that develops methods and
software tools for understanding biological data.
Bioinformatics is conceptualising biology in terms of molecules (in the
sense of physical chemistry) and applying "informatics techniques"
(derived from disciplines such as applied maths, computer science and
statistics) to understand and organise the information associated with
these molecules, on a large scale.
In short, bioinformatics is a management information system for
molecular biology and has many practical applications.
As an interdisciplinary field of science, bioinformatics combines computer
science, statistics, mathematics, and engineering to analyze and
interpret biological data.
Biological data are being produced at a phenomenal rate.

Bioinformatics
For example as of years before, the GenBank repository of nucleic
acid sequences contained 8,214,000 entries and the SWISS-PROT
database of protein sequences contained 88,166.
On average, these databases are doubling in size every 15 months .
 In addition, since the publication of the H. influenzae genome,
complete sequences for over 40 organisms have been released,
ranging from 450 genes to over 100,000.
Add to this the data from the myriad of related projects that study
gene expression, determine the protein structures encoded by the
genes, and detail how these products interact with one another, and
we can begin to imagine the enormous quantity and variety of
information that is being produced.

Bioinformatics
As a result of this surge in data, computers have become
indispensable to biological research.
Such an approach is ideal because of the ease with which computers
can handle large quantities of data and probe the complex dynamics
observed in nature.
Bioinformatics, the subject of the current discussion, is often defined
as the application of computational techniques to understand and
organise the information associated with biological macromolecules.
This unexpected union between the two subjects is largely attributed
to the fact that life itself is an information technology; an organism’s
physiology is largely determined by its genes, which at its most basic
can be viewed as digital information.

Bioinformatics
At the same time, there have been major advances in the
technologies that supply the initial data; Anthony Kerlavage of
Celera recently cited that an experimental laboratory can produce
over 100 gigabytes of data a day with ease.
This incredible processing power has been matched by
developments in computer technology; the most important areas of
improvements have been in the CPU, disk storage and Internet,
allowing faster computations, better data storage and
revolutionalised the methods for accessing and exchanging data.

Bioinformatics
The aims of bioinformatics are threefold.
First, at its simplest bioinformatics organises data in a way that allows
researchers to access existing information and to submit new entries as
they are produced, eg the Protein Data Bank for 3D macromolecular
structures.
While data-curation is an essential task, the information stored in these
databases is essentially useless until analysed.
Thus the purpose of bioinformatics extends much further.
The second aim is to develop tools and resources that aid in the
analysis of data.
For example, having sequenced a particular protein, it is of interest to
compare it with previously characterised sequences.

Bioinformatics
This needs more than just a simple text-based search and programs
such as FASTA and PSI-BLAST must consider what comprises a
biologically significant match.
Development of such resources dictates expertise in computational
theory as well as a thorough understanding of biology.
The third aim is to use these tools to analyse the data and interpret
the results in a biologically meaningful manner.
Traditionally, biological studies examined individual systems in
detail, and frequently compared them with a few that are related.
 In bioinformatics, we can now conduct global analyses of all the
available data with the aim of uncovering common principles that
apply across many systems and highlight novel features.

Tables showing Sources of data used in bioinformatics, the quantity of each type of
data that is currently available, and bioinformatics subject areas that utilise this data.
Data source Data size Bioinformatics topics
Raw DNA sequence 8.2 million
sequences (9.5
billion bases)
Separating coding and non-coding regions Identification of
introns and exons Gene product prediction Forensic analysis
Protein sequence 300,000 sequences
(~300 amino acids
each)
Sequence comparison algorithms Multiple sequence
alignments algorithms Identification of conserved sequence
motifs
Macromolecular
structure
13,000 structures
(~1,000 atomic
coordinates each)
Secondary, tertiary structure prediction 3D structural
alignment algorithms Protein geometry measurements
Surface and volume shape calculations Intermolecular
interactions
Genomes 40 complete
genomes (1.6
million – 3 billion
bases each)
Characterisation of repeats Structural assignments to genes
Phylogenetic analysis Genomic-scale censuses
(characterisation of protein content, metabolic pathways)
Linkage analysis relating specific genes to diseases
Gene expression largest: ~20 time
point measurements
for ~6,000 genes
Correlating expression patterns Mapping expression data to
sequence, structural and biochemical data
Other data
Literature 11 million citations Digital libraries for automated bibliographical searches
Knowledge databases of data from literature
Metabolic pathways Pathway simulations

Bioinformatics
Scientific euphoria has recently centered on whole genome
sequencing.
As with the raw DNA sequences, genomes consist of strings of
base letters, ranging from 1.6 million bases in Haemophilus
influenzae to 3 billion in humans.
An important aspect of complete genomes is the distinction
between coding regions and noncoding regions –'junk' repetitive
sequences making up the bulk of base sequences especially in
eukaryotes.
We can now measure expression levels of almost every gene in a
given cell on a whole-genome level although public availability of
such data is still limited

Bioinformatics
Nucleotide and Genome sequences:
The biggest excitement currently lies with the availability of complete
genome sequences for different organisms.
The GenBank, EMBL (European Molecular Biology Laboratory) and
DDBJ (DNA Data Bank of Japan) databases contain DNA sequences for
individual genes that encode protein and RNA products.
 Much like the composite protein sequence database, the Entrez
nucleotide database compiles sequence data from these primary
databases.
The Entrez genome database brings together all complete and partial
genomes in a single location and currently represents over 1,000
organisms.

Bioinformatics
Protein sequence databases:
Protein sequence databases are categorized as primary, composite or
secondary.
Primary databases contain over 300,000 protein sequences and
function as a repository for the raw data.
Some more common repositories, such as SWISS-PROT and PIR
International , annotate the sequences as well as describe the
proteins’ functions, its domain structure and post-translational
modifications.

Bioinformatics
Structural databases:
Next we look at databases of macromolecular structures.
The Protein Data Bank, PDB, provides a primary archive of all
3D structures for macromolecules such as proteins, RNA, DNA
and various complexes.
Most of the ~13,000 structures are solved by x-ray
crystallography and NMR (Nuclear magnetic resonance), but some
theoretical models are also included.

Bioinformatics
Finding Homologues:
As described earlier, one of the driving forces behind bioinformatics
is the search for similarities between different biomolecules.
Apart from enabling systematic organisation of data, identification
of protein homologues has some direct practical uses.
The most obvious is transferring information between related
proteins.
For example, given a poorly characterised protein, it is possible to
search for homologues that are better understood and with caution,
apply some of the knowledge of the latter to the former.

Bioinformatics
Rational Drug Design.
One of the earliest medical applications of bioinformatics has been
in aiding rational drug design.
MLH1 is a human gene encoding a mismatch repair protein
(MMR) situated on the short arm of chromosome 3.
Through linkage analysis and its similarity to MMR genes in mice,
the gene has been implicated in nonpolyposis colorectal cancer.
Given the nucleotide sequence, the probable amino acid sequence
of the encoded protein can be determined using translation
software.

Bioinformatics
Further applications in medical sciences.
 Most recent applications in the medical sciences have centred on
gene expression analysis.
This usually involves compiling expression data for cells affected
by different diseases, e.g. cancer and ateriosclerosis, and comparing
the measurements against normal expression levels.
 Identification of genes that are expressed differently in affected
cells provides a basis for explaining the causes of illnesses and
highlights potential drug targets

Bioinformatics
With the current deluge of data, computational methods have
become indispensable to biological investigations.
Originally developed for the analysis of biological sequences,
bioinformatics now encompasses a wide range of subject areas
including structural biology, genomics and gene expression studies.
Two principal approaches underpin all studies in bioinformatics.
First is that of comparing and grouping the data according to
biologically meaningful similarities and second, that of analyzing
one type of data to infer and understand the observations for another
type of data.

PRIMER DESIGN
A primer is a strand of short nucleic acid sequences that serves as
a starting point for DNA synthesis.
The first thing for primer design is to decide on the gene you want
work with (protein, nucleotide sequence of interest).
You should thoroughly read many articles concerning the protein to
find important parts that protein (amino acid) e.g. antigenic site,
glycosylation site, receptor binding site, catalytic site depending on
the protein you are dealing with.
For example when working with Haemagglutinin (HA) of H1N1
subtype (2009 pandemic) the position (amino acid) that are important
for binding site to the receptor Y95 KGVTA133-137, W153, V155,
H183, S186, L194 and Q226.

PRIMER DESIGN
Other two binding sites which are more important are D190 and D225.
The catalytic site is PSIQSR 325-330. The glycosylation potential has
NX S or T and in H1N1 the position is 14, 26, 90, 279, 290, 484 and 543.
Following this thorough update; then go to start menu and type the
website e.g. www.ncbi.nlm.nih.gov for NCBI. Type the exact gene e.g.
Human influenza virus H1N1 neuraminidase gene, polA gene from E.
coli or any other microbial gene.
For microorganisms that their vaccine strain are available is better to
start with it to serves as a guide which may be available in the WHO or
CDC sites.
Then FAST (copy on FAST) copy and Wright click paste on TEXT
DOCUMENT → than save.

PRIMER DESIGN
After copying different sequence → then go to BioEdit →
open.
The file you have already saved will open.
For alignment → mark the sequences you want align → click
Accessory Application → click ClustalW Multiple alignment
→ click run ClustalW.
It will automatically align the sequences.

PRIMER DESIGN
To cut improper arranged ends (that is at the beginning and the end):
Select the site by clicking on the mode edit → then high light the end
you want to delete e.g. beginning or end.
Then press the backspace key in your computer.
This will delete the selected site.
Then look for the conserved region in the sequence before and after the
site you want amplified.
It is advisable to go back to atleast 100 nucleotide before and after the
region you want amplify.
This is in case that after finding the positive sample you will go ahead

PRIMER DESIGN
Note:
the nucleotides for the primer should be atleast 18-25nt. Though it
can be up to 30nt.
At the region of the primer should have atleast 1 or 2 not more
3̕
than 3 C or G.
The CG content should be between 40-65% in the primer.
This is to allow the differences between the annealing temperatures
to be 4-5°C below the melting temperature.
The melting temperature should be 55-75°C and the annealing
should be 4-5°C below as stated above.
The differences in melting temperature between Forward and
Reverse should not be more than 2-3°C and at most 5°C.

PRIMER DESIGN
To check for the melting temperature, CG % content and the
nucleotide length
select positions of the nucleotide → go to edit and copy → go to
sequence → move to PCR primers/Oligo. Then go to calc. TM’s.
Then paste the nucleotide sequence without changing the default of
the program → then press calc. in the BioEdit.
These parameters can also be obtain using Oligo 7 software.
For TM, ABI Tm calculator can also be used and it produces more
accurate Tm reading for the nucleotide sequence.

PRIMER DESIGN
USING OLIGO 7 SOFTWARE IN PRIMER DESIGNING
PROCESS
If you want check primer with Oligo 7 you should copy the primer
sequence and go to Oligo → click file → new → edit → paste →
press accept.
It will automatically display a graph for melting temperature (Tm)
that shows different Tm reading.
Then go to analyse → then pick one by one (that is Duplex
formation, Hairpin formation, Composition and Tm (optional – it is
better to concentrate on the first two listed above) and False priming
Site (optional).

PRIMER DESIGN
For Example: Duplex formation you should take note the following
∆G should be negative
Tm should be lower than that of the primer (e.g. 10-20°C lower than the primer
Tm).
If there is any loop - it should not be at the region (although if unpreventable
3̕
loop is observed; the ∆G should be really negative → this will allow you to accept
it if there is no chance of moving or changing the selected primer sequence).
If there is chance, it is better to have perfect primer without a loop or hairpin.
Note: This applies to both Forward and Reverse primers. Reverse complement the
sequence of the Reverse primer.
The temperature (Tm) of F and R should be almost the same with difference not
more than 4°C in between.

PRIMER DESIGN
Now you should go to NCBI for nucleotide BLAST.
Copy the primer sequence designed and paste in the first box of the
nucleotide sequence “blast n” → then give the title of the primer in the job title
Colum/box → go down to “Others” click.
Then press the BLAST bottom. Just wait for some seconds; it will
automatically display the result.
Move down to check the name of the gene or sequence that the primer can
attach to.
Then down again to be sure of the sequence to which the primer attached to.
This BLAST should be done for both F and R primers one by one and for both
of them at the same time. Just like this: F…………2n R.
 F-ATGAAGGCAATACTAGTAGTTCTGCnnnnnnnnnnnnnnnnnR-
CAATGGAAGAAATGCTGGATCT.

PRIMER DESIGN
For BLAST in the Strand:
The Forward and Reverse primer should be the same strand (that is
for this you should make the Reverse primer to exactly the same as
copy from the original nucleotide sequence).
But for sending to the company for primer synthesis it should be
send in a Reverse Complement form.

PRIMER DESIGN
OLIGO-7.0 IN ANOTHER FASSION
When you get different sequences from different research including
the vaccine or any other reference sequence for a particular
organism → then copy it and Open Oligo 7.0 program and paste.
Then click accept → go to search for primer and probe click on it.
Then click on Ranges and change the length of PCR product if you
want.

PRIMER DESIGN
Press OK and then search.
 It will give you the position of F and R.
Choice one and click on it. It will give you the Tm and the position
of the NS (nucleotide sequence).
 For more information go to “analyse” as previous → then key
information → selected primers click on it, it will give you the
sequence, Tm and other information.

PHYLOGENETIC TREE
It is a tree that shows relationship between different isolate of an
organism circulating in an environment.
After multiple alignment of all your research sequences and other
sequences from previous study (including vaccine strain if
available) and cutting the unnecessary ends save in Phylip4
format.
There are two type of phylogenetic tree:
Rooted tree
Unrooted tree

PHYLOGENETIC TREE
Firstly, Open Tricon → Distance estimation and click.
Click start distance estimation
Select the file saved already with the sequence → select Philip
interleaved → click Okay.
Then choice the method which is usually “Kimura” for example
in case of Influenza and Measles.
Then Bootstrap analysis → click yes → then click Okay.
After completion click Okay.

PHYLOGENETIC TREE
Secondly, Infer tree Topology
Click start → then it will ask again and click yes.
The method usually selected is Neighbor joining →Okay.
Then Finish Okay.

PHYLOGENETIC TREE
Thirdly Root Unrooted Tree
Start rooting unrooted tree by clicking on the menu → Bootstrap
analysis → yes.
Select single sequence (Forced) then OK → then choose the root
e.g. California →Ok.

PHYLOGENETIC TREE
Fourthly then Draw phylogenetic Tree
Go to file →Open → new tree.
Select the one you want and keep on clicking until it give you the
desire tree shape you want.
Go to Bootstrap values then click on it.
Choose the % you want (it is better to choose 70% and above).
Also choose the position of the number where to be place then click
Ok.
Go to distance scale?

PHYLOGENETIC TREE
Note: you select group and to explain the size of the font, the
color and symbol (that is regular or not).
Then click Ok.
To print the tree, → pdf creator and save on the dextop and save
which can later be use in article.
 For saving → save tree as a Tricon file → then give name and
save.
Then for you to open the saved Tricon tree you have to go to
TRICON TREE WINDOW → click it and then click on Draw
phylogenetic tree and go to file → open →tree in Tricon format
click and choose and open.

RT-PCR
The invention of polymerase chain reaction (PCR) has been a
milestone in the history of biological and medical sciences.
 RT-PCR stands for Reverse Transcription-Polymerase Chain
Reaction.
It is a technique used in molecular biology studies that allows the
detection and quantification of mRNA.
It is a very sensitive method that shows whether or not a specific
gene is being expressed in a given sample.
RT-PCR is used to locate and quantify known sequences of mRNA in
a sample.
The first step in RT-PCR uses reverse transcriptase and a primer to

RT-PCR
If the mRNA is present, the reverse transcriptase and primer will
anneal to the mRNA sequence and transcribe a complimentary strand
of DNA.
This strand is then replicated with primers and Taq Polymerase, and
the standard PCR protocol is followed.
This protocol copies the single stranded DNA millions of times in a
small amount of time to produce a significant amount of DNA.
 The PCR products (the DNA strands) are then separated with
agarose gel electrophoresis.
If a band shows up for the desired molecular weight, then the
mRNA was in fact present in the sample, and the associated gene
was being expressed.

RT-PCR
The conventional PCR led to the emergence of RT-PCR, qPCR and
combined RT-PCR/q-PCR.
PCR has also enabled the successful completion of the ‘human genome
project’ by enabling the amplification and sequencing of the human
genes, which has further laid the foundation of genetic engineering and
has now even made it possible to make useful changes in the genome of
an organism.
The results from RT-PCR are used in two main ways.
First, RT-PCR shows us whether or not a specific gene is being
expressed in a sample.
If a gene is expressed, its mRNA product will be produced, and an
associated band will appear in the final agarose gel with the correct
molecular weight for the gene.

Principles
In RT-PCR, the RNA template is first converted into
a complementary DNA (cDNA) using a reverse transcriptase. The
cDNA is then used as a template for exponential amplification using
PCR.
The principle of PCR is based on the fact that at high denaturing
temperatures nearing 95°C, the two strands of the target DNA molecule
separate due to breaking of A-T and G-C bonds.
At the annealing temperatures in the range of 50-65°C, the
complimentary forward and reverse primers bind at the 3’ end of the
flanking regions of the separated single stranded target DNA molecule.
The Taq polymerase then extends the new DNA strand by adding
dNTPs and the double stranded molecule restructures itself at the
extension temperature of 72°C.

Essential Components of Standard PCR
Component of reaction mixture:
Taq/other thermostable polymerases
Template DNA
Primers
dNTPs
MgCl2
Autopipettes / Plasticwares /Gloves
Thermal cycler

One-step RT-PCR vs two-step RT-PCR
The quantification of mRNA using RT-PCR can be achieved as
either a one-step or a two-step reaction.
The difference between the two approaches lies in the number of
tubes used when performing the procedure.
In the one-step approach, the entire reaction from cDNA synthesis
to PCR amplification occurs in a single tube.

One-step RT-PCR vs two-step RT-PCR Cont..
On the other hand, the two-step reaction requires that the reverse
transcriptase reaction and PCR amplification be performed in
separate tubes.
The one-step approach is thought to minimize experimental
variation by containing all of the enzymatic reactions in a single
environment.
However, the starting RNA templates are prone to degradation in
the one-step approach, and the use of this approach is not
recommended when repeated assays from the same sample is
required.
Additionally, one-step approach is reported to be less accurate
compared to the two-step approach.

One-step RT-PCR vs two-step RT-PCR Cont..
It is also the preferred method of analysis when using DNA binding
dyes such as SYBR Green since the elimination of primer-dimers
can be achieved through a simple change in the melting temperature.
The disadvantage of the two-step approach is susceptibility to
contamination due to more frequent sample handling.

Procedure
Initial denaturation occurs at 90-95°C for 3-5 minutes, where the
two strands of the double stranded target DNA molecule separate.
Initial denaturation is followed by 30-35 cycles of denaturation,
annealing and extension.
The number of PCR cycles depends on the amount of template DNA
in the reaction mix and on the expected yield of the PCR product.
Denaturation involves heating the double stranded target DNA
molecule at 90-95°C for 30-55 seconds.
Annealing step allows binding of the complimentary forward and
reverse primers to the 3’ flanking regions at 50-65°C for 30-55
seconds.

Procedure
Extension step occurs at 72°C for 30-55 seconds by adding
complimentary dNTPs to the new strands.
After the last cycle, the samples are usually incubated at 72°C for
5-15 minutes to fill-in the protruding ends of newly synthesized
PCR products.
Holding or storage of the PCR products at 4°C for infinity.

Post amplification detection
The amplified PCR product is observed as a fluorescent pink band
by agarose gel electrophoresis following ultraviolet trans
illumination of the agarose gel stained in Ethidium bromide (EtBr)
solution.
EtBr has been used since many years for the visualization of
nucleic acids in agarose gel.
EtBr is also a potent mutagen, causing mutations in the living cell.
Therefore, gels should be disposed in an appropriately labelled
hazardous waste container with date and handed over to the
Environmental Health & Safety (EHS) department

Types of PCR
Standard PCR- Variants
Reverse Transcription-PCR (RT- PCR)
Real time-PCR or quantitative PCR (qPCR)
RT-PCR/qPCR combined

Types of PCR
Standard PCR- Variants
The modifications in the basic technique of PCR led to the development
of variants in PCR that are described below:
1. Allele specific PCR (Tetra-primer ARMS PCR)
 Allele specific PCR allows direct detection of point mutation in
DNA.
 This technique requires prior knowledge of the target DNA sequence
such as differences between alleles and utilises the primer with 3’
mismatch ends encompassing the single nucleotide variations.
 Two allele specific primers, one for each allele of the SNP are
required, which contain one of two polymorphic nucleotides at the 3'
end.

Types of PCR
2. Asymmetric PCR
This variation of PCR is used to preferentially amplify only one strand
of the target DNA molecule by using unequal primer concentrations, as
such replication occurs arithmetically by using the excess primer.
3. Colony PCR
It is a type of PCR routinely used in bacterial genomic studies.
Insertion of high copy number plasmids such as pUC 18, pUC 19 or
pBluescript in bacteria is routinely performed for a variety of purposes
and colony PCR quickly screen these plasmid inserts.
It has several advantages over the traditional methods of blue/white
screening as it can determine both the insert size and orientation in the
vector

Types of PCR
4. Degenerate PCR
It is a variant of PCR which employs degenerate primers to amplify unknown
sequences of DNA, related to a known DNA sequence.
Degenerate primers are designed on the basis of known and sequenced gene
homologs.
This technique allows identification of new members of a gene family or
orthologous genes from different organisms.
5. Hotstart PCR
This technique involves steps of the conventional PCR, except that the Taq
polymerase is added after the rest of the PCR components are heated to the
DNA melting temperature, so as to avoid non-specific amplification at lower
temperatures.
Alternatively, covalently bound inhibitors that dissociate from Taq polymerase
only after reaching the Tm can also be added

Types of PCR
6. Inverse PCR
Whereas conventional PCR requires complimentary primer pair for both the 3’
ends of the target DNA, Inverse PCR allows amplification of DNA with only one
known sequence.
This technique requires a sequence of restriction digestions and ligations which
result in the formation of a looped DNA fragment which can further be primed
from a section of known DNA sequence for PCR.
7. Miniprimer PCR
The standard PCR methods require Taq polymerase whose efficiency of DNA
synthesis is less than other replicative enzymes due to their longer primers (20-30
nucleotides) requirement.
Therefore, new PCR method has been developed called miniprimer PCR.
In this PCR, engineered Taq polymerase and 10 nucleotides long ‘miniprimers’ are
used.

Types of PCR
8. Multiplex PCR
Multiplex PCR is a modification of PCR in order to rapidly detect deletions or
duplications in a large gene.
In 1988, deletions in the dystrophin gene were first detected by multiplex-PCR
method.
Multiplex- PCR mix makes use of multiple primer sets within a single PCR
mixture to produce amplicons of varying sizes which are specific for different
sequences of DNA.
This variant of PCR, targets multiple genes at once in a single test run which
would otherwise require several times the reagents and more time to perform.
The base pair length of the amplicons, should be different enough to segregate
well and form distinct bands when visualized by gel electrophoresis.
Multiplex- PCR has been successfully applied in many areas of DNA testing such
as analysis of deletions, mutations and polymorphisms, microsatellites and SNPs

Types of PCR
9. Nested PCR
It is a modification of PCR designed to minimize the amplification
of non-specific and spurious PCR products, which may result due to
primer binding at unexpected or unwanted sites similar to the target
DNA.
Nested PCR involves 2 sets of primers which are utilized in two
successive runs of the PCR reaction.
The second set of primers functions to bind to a secondary target
within the sequence amplified by the first set of primers, as it is
highly unlikely that the spurious or unwanted sequence will have
binding site for both the sets of primers

Types of PCR
10. Touchdown PCR
This technique enables ruling out the amplification of non-specific
sequences by using early steps of PCR cycles at high temperatures
and with subsequent cycles, the annealing temperatures are
decreased in increments.
This allows specific primer to anneal at the highest temperature
that is least permissive for non-specific binding and generates only
the sequence of interest.
11. Reverse Transcription-PCR (RT- PCR)
This technique enables quantitative detection of levels of RNA
expression by creating complimentary DNA (cDNA) from RNA
with the help of reverse transcriptase, followed by further
amplification of cDNA using standard PCR

11. Reverse Transcription-PCR (RT- PCR) Cont..
Howard Temin from the University of Wisconsin–Madison made
the discovery of reverse transcriptases in RSV (Rous Sarcoma Virus),
which were then later independently isolated by David Baltimore in
1970 from two RNA tumour viruses: R-MLV (Rauscher- Murine
Leukemia Virus) and RSV.
Both shared the 1975 Nobel Prize in Reverse transcriptase enzyme
includes an RNA-dependent DNA polymerase, a DNA- dependent
DNA polymerase and ribonuclease H activity which work in sync to
perform transcription.
The retroviral reverse transcriptases, including Avian
Myeloblastosis Virus (AMV) and Moloney murine leukemia virus
(MMLV) are the most characterised reverse transcriptases used in the
field of molecular biology.

Types of PCR Cont..
12. Real time-PCR or quantitative PCR (qPCR)
 qPCR introduced in 1992 by Higuchi and co-workers that enables
detection of fluorescent reporter dye, such as SYBR Green I to measure
the amplification of DNA at each cycle of PCR.
During the log linear phase of amplification, the fluorescence increases
to a point which becomes measurable and is called as the Threshold cycle
(CT) or Crossing point.
Therefore, by using serial dilutions of a known quantity of standard
DNA, the amount of DNA or cDNA of unknown sample can be
calculated as CT value by plotting a standard curve of log concentration
vs CT.
qPCR combines the amplification and detection into a single step
thereby eliminating the need for any post amplification processing of the
sample.
The other advantages of qPCR are sensitivity, real time detection of
reaction progress, speed of analysis and precise measurement of the
examined material in the sample.

Types of PCR Cont..
13. RT-PCR/qPCR combined
In case of qualitative detection of RNA expression, reverse
transcription (RT-PCR) polymerase chain reaction technique is used
through conversion of RNA template to cDNA where as for
quantitative detection of RNA expression, both RT-PCR and qPCR
techniques are merged and this combined technique is called qRT-
PCR/ quantitative RT-PCR or RT-qPCR.

Advantages of PCR Technique
Since amplification is carried out by designing the complementary
primers, the technique is highly specific.
It is relatively fast enough generating a billion copies of
amplification in less than three hours.
Based on the type of genetic material (DNA or RNA) suitable
modifications can be made easily and the technique can be easily
used for a wide range of applications in almost all sorts of organisms
ranging from microorganisms to plant and animal kingdom.

Disadvantages of PCR Technique
The first and foremost drawback is its cost. It is an expensive technique
in comparison to the conventional tests.
Performing a PCR require a great degree of skill and expertise.
Furthermore, to carry out the PCR one must have a sound knowledge of
the bioinformatics to design primers, to incorporate restriction sites.
The technique is available in only those labs that have specialized
molecular biology testing and analysis techniques.
Most of the times nucleic acid from non-viable organisms is also
amplified along with the desired samples.
The analysis of samples after PCR exposes a researcher to harmful
chemicals like EtBr, dyes, fluorochromes and UV light which are
carcinogenic

Applications of PCR
Forensic science
 PCR is an important tool in DNA profiling, fingerprinting, DNA
typing and DNA testing.
This technique enables identification of one person among millions
of others.
Samples of DNA extracted from crime scene can be compared with
DNA of suspects or DNA database.
Also DNA fingerprinting enables parental testing to identify
biological parentage of a child.

Applications of PCR
Medicine and diagnostics
 Prospective parents can be subjected to gene testing for the presence
of genetic diseases and hence the probability of children being carriers
of the same can be ascertained.
Prenatal testing can be performed by amniocentesis, chronic villus
sampling or fetal cells circulating in mother’s blood to ascertain the
possibility of mutations in the embryo.
Tissue-typing can be done prior to performing organ transplantation by
using PCR for checking compatibility between donor and recipient.
This method has replaced the traditional antibody based blood type test
for identifying antigens on the surface of the body cells and tissues.
Therapy regimens can be customised for individual patients by using
PCR based tests to study mutations in oncogenes in certain forms of
cancer.

Applications of PCR
Since antibodies to HIV do not appear until many weeks after infection, PCR
based tests have been developed that enable detection of even a single viral
genome among the host cells.
Similarly, donated blood, newborns and effects of antiviral treatments can be
done immediately.
Moreover, donated blood can also be tested for bacterial contamination using
real-time PCR.
In case of Tuberculosis, which otherwise requires sputum sample collection and
culture in laboratory, PCR based tests have enabled detection of both live and
dead microorganisms.
Moreover, detailed gene analysis enables detection of antibiotic resistance as
well as effects of therapy.
PCR based testing has enabled detection of spread of infectious microorganisms
in domestic or wild animals

Applications of PCR in Summary
Molecular Identification
Sequencing
Genetic Engineering
 Molecular Archaeology
Molecular Epidemiology
 Molecular Ecology
DNA fingerprinting
Classification of organisms
Genotyping
 Pre-natal diagnosis

Applications of PCR in Summary
Mutation screening
Drug discovery
Genetic matching
Detection of pathogens
Bioinformatics
Genomic cloning
Human Genome Project
Site-directed mutagenesis

BLOTTING
Blotting refers to the technique, where molecules (nucleic acids or
proteins) that have been separated by electrophoresis are transferred
or blotted onto a specific type of paper usually nitrocellulose or
PVDF (polyvinyl difluoride) by the application of an electrical
current (electroblotting).
Blotting have been developed to be highly specific and sensitive
and have become important tools in both molecular biology and
clinical research.
The blotting methods are fairly simple and usually consist of four
separate steps:
electrophoretic separation of protein or of nucleic acid fragments
in the sample;
transfer to and immobilization on paper support;
binding of analytical probe to target molecule on paper;

General principle
Molecules in a sample are first separated by electrophoresis and
then transferred on to an easily handled support medium or
membrane.
This immobilizes the protein or DNA fragments, provides a
faithful replica of the original separation, and facilitates subsequent
biochemical analysis.
After being transferred to the support medium the immobilized
protein or nucleic acid fragment is localized by the use of probes,
such as antibodies or DNA, that specifically bind to the molecule of
interest.
Finally, the position of the probe that is bound to the immobilized
target molecule is visualized usually by autoradiography.

Blotting Techniques
There are three main blotting techniques that have been
developed and are commonly called
Southern
northern
western blotting.
Southern blot
Southern blot is a method used to check for the presence of a
DNA sequence in a DNA sample.
The method is named after its inventor, the British biologist
Edwin Southern.

Blotting Techniques
Northern blot
The northern blot technique is used to study gene expression by
detection of RNA (or isolated mRNA) in a sample.
With northern blotting it is possible to observe cellular control over
structure and function by determining the particular gene expression
levels during differentiation, morphogenesis, as well as abnormal or
diseased conditions.
This technique was developed in 1977 by James Alwine, David Kemp
and George Stark at Stanford University.
Northern blotting takes its name from its similarity to the first blotting
technique, the Southern blot.
The major difference is that RNA, rather than DNA, is analyzed in the
northern blot.

Northern blot Applications
Northern blotting allows in observing a particular gene's expression
pattern between tissues, organs, developmental stages,
environmental stress levels, pathogen infection.
The technique has been used to show over expression of oncogenes
and down regulation of tumor-suppressor genes in cancerous cells
when compared to 'normal' tissue, as well as the gene expression in
the rejection of transplanted organs.

Western blot
The western blot (alternatively, immunoblot) is used to detect
specific proteins in a given sample of tissue homogenate or
extract.
The method originated from the laboratory of George Stark at
Stanford. The name western blot was given to the technique by W.
Neal Burnette.

Eastern blotting
It is a technique to detect protein post translational modification and is an
extension of the biochemical technique of western blotting.
Proteins blotted from two dimensional SDS-PAGE gel on to a PVDF or
nitrocellulose membrane are analyzed for post-translational protein modifications
using probes specifically designed to detect lipids, carbohydrate, phosphomoieties
or any other protein modification.
The technique was developed to detect protein modifications in two species of
Ehrlichia- E. muris and IOE.
Cholera toxin B subunit (which detects lipids), Concanavalin A (which detects
glucose moieties) and nitrophospho molybdate-methyl green (detects
phosphoproteins) were used to detect protein modifications.
The technique showed that the antigenic proteins of the non-virulent E. muris are
more post-translationally modified than the highly virulent IOE.
The technique was conceptualized by S. Thomas

Applications of Blotting Techniques
1. Southern blotting technique is widely used to find specific
nucleic acid sequence present in different plant species.
2. Northern blotting technique is widely used to find gene
expression and regulation of specific genes.
3. By using blotting technique we can identify infectious agents
present in the sample.
4. We can identify inherited disease.
5. It can be applied to mapping restriction sites in single copy gene.

Disadvantages of Blotting Techniques
1. The process is a complex, cumbersome and time consuming one.
2. It requires electrophoretic separation.
3. Only one gene or RNA can be analysed at a time.
4. Gives information about presence of DNA, RNA or proteins but
does not give information about regulation and gene interaction.

Tissue Culture
It is a method of biological research in which fragments of tissue from an animal or
plant are transferred to an artificial environment in which they can continue to
survive and function.
The cultured tissue may consist of a single cell, a population of cells, or a whole or
part of an organ.
Cells in culture may multiply; change size, form, or function; exhibit specialized
activity (muscle cells, for example, may contract); or interact with other cells.
Cells may be grown in a culture medium of biological origin such as blood
serum or tissue extract, in a chemically defined synthetic medium, or in a
mixture of the two.
A medium must contain proper proportions of the necessary nutrients for the
cells to be studied and must be appropriately acid or alkaline.
Cultures are usually grown either as single layers of cells on a glass or plastic
surface or as a suspension in a liquid or semisolid medium.

Tissue Culture Cont..
To initiate a culture, a tiny sample of the tissue is dispersed on or in
the medium, and the flask, tube, or plate containing the culture is then
incubated, usually at a temperature close to that of the tissue’s normal
environment.
Sterile conditions are maintained to prevent contamination with
microorganisms.
Cultures are sometimes started from single cells, resulting in the
production of uniform biological populations called clones.
Single cells typically give rise to colonies within 10 to 14 days of
being placed under culture conditions.

Primary cultures and established cell lines
There are two main types of cultures: primary (mortal) cultures and
cultures of established (immortal) cell lines.
 Primary cultures consist of normal cells, tissues, or organs that are
excised directly from tissue collected by biopsy from a living
organism.
Primary cultures are advantageous in that they essentially model the
natural function of the cell, tissue, or organ under study.
However, the longer the samples are maintained in culture, the more
mutations they accumulate, which can lead to changes in
chromosome structure and cell function.

In addition, primary cultures generally are mortal.
Cells undergo an aging process whereby they multiply for only 50
to 100 generations, after which the rate decreases markedly.
The point at which cells in primary cultures stop growing, or
undergo replicative senescence, marks the so-called Hayflick limit
(named for its discoverer, American microbiologist Leonard
Hayflick).
By contrast, established cell lines can be perpetuated indefinitely.
Such cell lines generally are derived from tumour biopsies from
patients, or they may be generated from primary cells that have
undergone mutations that enabled them to overcome the Hayflick
limit and continue replicating.

Cells in established lines accumulate mutations over time that can change their
character.
Processing of cultured cells and tissues
Live cultures may be examined directly with a microscope, or they may be
observed by means of photographs and motion pictures taken through the
microscope.
Cells, tissues, and organs may also be killed, fixed (preserved), and stained for
further examination.
Following fixation, samples can also be embedded (e.g., in a resin) and cut
into thin sections to disclose additional details under a light or electron
microscope.
Cells in tissue culture are subjected to a broad range of experimental treatment.
For example, viruses, drugs, hormones, vitamins, disease-
causing microorganisms, or suspected cancer-producing chemicals may be
added to the culture.
Scientists then observe the cells, looking for global changes in cell behaviour
or function or for changes in specific molecules, such as alterations in the
expression of a particular protein or gene.

Tissue Culture Media
The commonly used media include RMPI-1640, Viral transport medium
(VTM), Other media types are sometimes used for specific applications for
example SFM (Life Tech) for growing hybridomas for antibody purification;
DMEM for some mouse lines; Iscoves for chicken cell culture.
Media Supplements
Medium is routinely supplemented before use in culture by adding 25 or 50
mL of FBS (fetal bovine serum, CSL) ie 5% or 10% depending on culture
needs (can be kept as a frozen stock in aliquots).
Glutamine is added to 2mM since this essential amino acid is not stable in
aqueous solutions. It is usually added as 5mL of a 200mM solution (5g in
170mL water). Glutamax™ is now used which does not break down (both
can be kept as a frozen stock in aliquots).
Other supplements are used for specific culture situations such as for
transfected cell lines or for Fetal Thymic Organ Culture (FTOC)

Thawing cell lines from liquid nitrogen stocks
Aspirate 8-9mL of media into a 10 mL tube and place this on ice or
in a refrigerator for 15 min before thawing cells -the preservative
DMSO (dimethyl sulfoxide, BDH/Merck) if allowed to warm around
cells may enter them and become toxic.
Bring vial(s) from nitrogen store immediately to a 37°C water bath.
If thawing a number of vials store them in dry ice prior to thawing
and thaw only one or two vials at a time.
Swirl a vial or two in the bath taking care not to immerse
completely, to prevent any bath water from entering the sealed cap.
When there is only a small amount of ice left in the tube ie still cold,
take the vial(s) to a sterile cabinet/hood and coat with 70% ethanol to
wash off bath water and kill any bacteria on the outside surfaces.

Thawing cell lines from liquid nitrogen stocks
Aspirate the contents of the vial(s) (usually 1-2 mL) into the 8-9 mL
cold media and centrifuge immediately 400g, 5 min, 4°C.
Wash cells in fresh media by aspirating the supernatant,
resuspending the cells in fresh media and centrifuging again.
Finally resuspend the cells in 1 mL and count then plate.
Counting cells
Methods include ethidium bromide / acridine orange (EtBr /AcOr)
or Trypan Blue dye inclusion/exclusion, but others are also valid
such as the use of a Coulter™ counter or similar, though the latter do
not truly count viability.

Ethidium Bromide Acridine Orange Method
The cells suspension may need to be diluted to count a reasonable number
-1/10 to 1/50 is often sufficient.
Take 10 µL of the diluted cells and add this to 10µL of EtBr/AcOr and
mix gently.
Add 10µL to the edge of a haemocytometer coverslip mounted on the
haemocytometer chamber. Repeat on the other side.
Count at least 100 cells for accuracy and calculate the cell number / mL.
The main centre grid is 1mm x 1mm in area and 0.1 mm deep i.e. 10 -
4
mL e.g. Cell count x 2 x dilution (ie x 10 or x 50) x 10 4
= cells / mL
Total cells = cells/ml x the original volume of fluid from which the cell
sample was removed; % Cell viability = total viable cells (unstained)/total
cells x 100.

Plating cells
This term refers to decanting an appropriate cell suspension into tissue culture
vessels which may be of a tray type of various well numbers, flasks of varying
volumes or even petri dishes (where the term orginates)
Freezing cells down
Cells to be frozen are harvested from the culture vessel and centrifuged as before,
then resupsended in cold 10% DMSO in FBS(can be kept as a frozen stock in
aliquots)
For quantity, as a rule of thumb:
1 vial (1 mL of 10% DMSO in FBS) per small flask of cells -15 cm2
3 vials for a medium flask -75 cm2
6 vials for a large flask -150 cm2
The vials are quickly transferred to a 'Mr Frosty™' or if one is not available, a wadd
of cotton wool and placed at -70°C until frozen (wait at least 1-2 hours to O/N)
Then transfer the vials to liquid nitrogen storage containers.

Adherent vs Suspension culture
Cells that grow in suspension vs those that adhere to the tissue
culture vessel are handled differently.
Suspension cultures may be divided by simple pipetting /
resuspension.
Adherent cells actively bind to the plastic ware, usually in a
reversible fashion.
They may be split/divided/transferred by the use of EDTA alone or
solution containing EDTA and trypsin

Method
Remove/aspirate the media from the cell culture vessel
Wash the vessel/cells with serum free media or PBS to remove traces of
FBS containing media
Add enough trypsin/EDTA (Life Tech pre-made stock or 0.1% trypsin /
2mM EDTA in balanced salt solution, no Mg or Ca) solution to cover the
base surface area and transfer the vessel to 37°C incubator for 5 minutes.
Using the trypsin/EDTA solution, resuspend and wash the vessel floor
the aspirate the cells to a tube containing media for washing by
centrifugation.
After washing the cells may be counted or divided appropriately for
further culture, application ie staining procedure, lysis etc, or
resuspended in 10% DMSO/FBS for freezing

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