Introduction to
Molecular Biology
Dr. M. Ravichandran
Department of Medical Microbiology and Parasitology
PPSP, USM
Ext: 4592
mravic@[Link]
[Link]
History of Molecular Biology
Year Discovery Inventor
1866 Study of inheritance Gregor Mendel
1944 DNA is the genetic Avery
material
1953 Structure of DNA Watson and Crick
1961 Genetic code Nirenberg and
Khorana
1977 DNA sequencing Sanger
(enzymatic method)
1985 Polymerase chain Kary B. Mullis
reaction
1997 Dolly- Sheep cloning Ian Wilmut
2001 Human genome Venter- Celera
Genomics 2
Glossary
Purines: Adenine and Guanine
Pyrimidines: Cytosine and Thymine (Uracil in RNA)
A = T/ A = U G ≡C
Gene is a segment of DNA which codes for a protein or RNA
Codon is a triplet of 3 nucleotides that represents amino acids or terminal
signals
Start codon ATG (AUG in RNA)
Stop codon TAA, TAG, TGA ( UAA, UAG, UGA in RNA)
ATG CAA AAG AAA TTG CTC CCC ATC CTT ATC TGA
Met Gln Lys Lys Leu Leu Pro Ile Leu Ile ---
ÏStart Ï Stop
Open reading frame (ORF):DNA sequence contains series of
codons, which can be translatable into protein. Start codon Î
Stop codon
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Up stream, down stream
Transcription: synthesis of RNA using DNA as template
Translation: Synthesis of Protein using RNA as template
Biotechnology: A sub discipline of biology in which biochemistry
based process such as DNA technology are used for practical
purposes
cDNA: DNA molecule that results from reverse transcriptase
activity on RNA
AA: Amino acid, approximate weight of one AA = 110 Daltons
bp: base pair
1AA is coded by 3 bases of DNA (codon)
Kbp: Kilo base pair (1000 base pair)
Vector: cloning vehicle eg: plasmid
Plasmid Example: pNAME
Signal sequence: It is an AA sequence required for protein secretion
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Standard
Genetic
Code
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Prokaryotes vs Eukaryots
Prokaryotes Eukaryotes
Simple Complex
Genes grouped in Operon Genes have Introns, Exons,
Polycistronic-Multiple gene 5’cap and 3’ PolyA
No Post translational Monocistronic-Single gene
modifications Post translational
modifications
Eg: glycosylation
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Gene expression in Prokaryots
DNA replication
(DNA polymerase)
Promoter Gene
DNA
Transcription
(RNA polymerase)
mRNA
Translation
(protein synthesis)
Protein
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Gene expression in eukaryots
Promoter Exon Intron Exon Intron Exon
DNA
Transcription (RNA polymerase)
Primary mRNA transcript
AAAAAA
Removal of introns, splicing
Mature mRNA m7G AAAAAA
Translation
Protein
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Electrophoresis
How do you see of DNA
DNA?
By spectrophotometer: OD 260 nm
1OD = 50ug of dsDNA
33ug of ssDNA 9
40ug of RNA
Agarose gel electrophoresis
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Samples that have been mixed with the sample buffer is then
load into each well
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Electrophoresis of DNA
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Antisense molecules
Antisense molecule - RNA or DNA complementary to mRNA
Normal gene expression
Protein
mRNA
Clone desired DNA fragment or transfer antisense
DNA/RNA into the cell
Antisense molecule RNA or DNA
No Protein
|||||||||||||||||||
expression
mRNA
Application:
•Inhibition of HIV viral replication inside the infected cells
•Block the expression of Onchogenes Do you want to know about13RNAi
•Inhibition of tomato ripening gene in tomatoes
RNAi
RNAi – interference RNA, which
can be used to silence a
gene specifically
Gene silencing using RNAi can
be done by two
methods
1. Injecting gene specific
dsRNA
2. Transfecting a cell with
a plasmid containing
with the specific gene
sequence and two
opposed promoters
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RNA Interference (RNAi)
Double stranded RNA is
introduced into a cell
Double stranded RNA is
chopped into pieces by
an enzyme
Guide RNA
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RISC – RNA induced silencing complex
RNA Interference (RNAi)
The pieces “guide” a
set of proteins to the
normal mRNA
The normal mRNA is
clipped into pieces and
destroyed
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Mechanism of RNAi
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RNAi in zebra fish embryo
No RNA i
With RNAi
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Some Facts About Biotechnology
Over 200 million people worldwide are being benefited from
biotechnology drug products and vaccines.
Biotechnology is responsible for hundreds of medical diagnostic tests
that keep the blood supply safe from the AIDS virus and detect other
conditions early enough to be successfully treated.
Biotechnology foods such as longer lasting tomatoes and better-tasting
carrots and peppers are in the market.
Hundreds of biopesticides and other agricultural products are now
used to improve our food supply and to reduce our dependence on
conventional chemical pesticides.
Environmental biotechnology products make it possible to more
efficiently clean up hazardous waste without the use of caustic chemicals or
incineration.
DNA fingerprinting, a biotech process, has dramatically improved
criminal investigation and forensic medicine, as well as afforded significant
advances in anthropology and wildlife management. 20
Suggested readings
• Principles of Gene Manipulation- An
introduction to Genetic Engineering, [Link]
& S.B primrose
• Genetics: TA Brown
• Molecular Biology: David Freifelder
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Albert Einstein
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Questions and suggestions
Dr. M. Ravichandran
Department of Medical Microbiology and
Parasitology
PPSP, USM
Ext: 4592
mravic@[Link]
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