Download Human Genome

Computational Biology 2
Pawan Dhar
BII
Lecture 1
Introduction to terms, techniques
and concepts in molecular biology
Molecular biology - a primer
Human body has
100 trillion cells
each containing
3 billion pairs of
nucleotides.
xxxxxxxxxxxxx
A 6 foot human
has so much of
DNA that it can
travel to and fro,
from Earth to
Sun 600 times !!!
RNA polymerase (yellow)
attaches to a DNA, walks
down the DNA and creates
a copy of DNA into a strand
of messenger RNA (blue)
The mRNA strand floats
free and finds a ribosome
(green).
Ribosome churns out a
chain of amino acids
(purple)
Amino-acid chain folds into
the protein
Glossary
Genome
The entire genetic component of an organism
ORF
A series of codons starting with an initiation
codon and ending with a termination
(open reading frame)
codon
DNA sequencing The technique for determining the order
of nucleotides in a DNA molecule
DNA replication
Synthesis of a new copy of DNA molecule
DNA polymerase An enzyme that synthesizes DNA
Cloning
Inserting DNA fragment into cloning vector
and propagating recombinant vector
in a host organism
Question time
An ORF represents an open reading
frame. Is there a CRF too ?
(CRF = closed reading frame)
Vectors
Vector
Insert size
Lambda vector
Cosmid, fosmid
PI
BAC, PAC
YAC
Mega-YAC
18 kb
40 kb
125 kb
300 kb
600 kb
1400 kb
p=95%
(human genome)
532 500
240 000
77 000
32 000
16 000
6 850
The size of the library depends on the cloning vector
Restriction Enzymes
DNA cutting enzymes found in bacteria.
Example:
Hae III 5’ G G C C 3’
3’ C C G G 5’
Blunt ends
EcoRI
Sticky ends
5’ G A A T T C 3’
3’ C T T A A G 5’
DNA ligase joins the broken ends
n
i
RI n
o io
c
E ct
a
Restriction
map of
pBR322
Question time
A student repeatedly failed to cut a
plasmid with two different restriction
endonucleases, even though he was
using the enzymes in good condition
and the plasmid had sites for both.
Plasmids
1. Extrachromosomal circular DNA molecules
which are not part of the bacterial chromosome.
2. Size range: 1 - 200 kb
3. Carry functions advantageous to hosts e.g.,
(a) produce enzymes which degrade antibiotics
or heavy metals (b) produce restriction and
modifying enzymes.
4. Replication is coupled to host replication in both
stringent (1-2 copies / division) and relaxed
manners (10-200 copies / division).
How do you insert foreign
DNA into plasmid ?
• Cut plasmid on a unique restriction site
• Also cut the insert by the same restriction
enzyme (preferably)
• Incubate linearized plasmid with the
fragment to be inserted
• Check insertion by gel electrophoresis
Finding a
way into
plasmid’s
territory
Question time
Devise a strategy to ligate an
“insert” with the “linearized
plasmid” given that the insert
has a blunt end and plasmid
has a sticky end ?
Transformation
Insertion of recombinant plasmid into bacteria
1. Chemical: e.g., calcium phosphate mediated
2. Physical : e.g., electroporation, DNA gun
3. Viral: e.g., retroviruses
Bacteria a living
photocopy
machine
Producing
insulin
through
genetic
engineerin
g
Effect of plasmid size on
transformation efficiency
Molecule
size (kb)
2.0
3.2
4.3
12.5
20.0
39.0
54.0
% Maximum
probability
57
100
86
43
36
14
6
Question time
Given the task of sequencing
a large stretch of DNA, what
plasmid size would you choose
for transformation into bacteria
and why ?
Artificial
vehicle
acting
naturally
What is cloning ?
A procedure to generate large number
of copies of a single sequence of DNA
Properties
1. The host will accept foreign DNA and can still
complete its life cycle
2. Permit the host to divide normally and be
distinguished from non transformed members
of community
cDNA cloning
It is a method of obtaining a DNA copy of the
mRNA and making a library out of it.
STEPS
1. Bind oligo DT to the poly A tail of the mRNA
2. Add reverse transcriptase and make its cDNA copy
3. Digest mRNA and add poly C tail to 3’end of DNA
4. Add oligo dG and synthesize a complementary strand
5. Add dC’s to the 3’end of double stranded cDNA
6. Insert dC tailed DNA to dG tailed linearized plasmid
7. Transform E.coli cells
8. Use selection makers to identify transformed cells
Question time
Is there a way to prevent self-ligation
of linearized plasmid ? If No explain
why, if Yes explain how.
Creatin
g
genomi
cs
What is a genomic library ?
A genomic library contains DNA
fragments which when aligned
together represent the entire genome.
The major problem
Not all the fragments find their way
into plasmids !!!
Question time
What strategy would you use
to include the entire genome
into plasmid library
Chromosome walking
It is a technique for constructing
a clone contig by identifying
overlapping fragments of cloned
DNA
Contig: is a continuous set of
overlapping DNA sequences
Question time
What is the best way to
“walk through” a highly repetitive
DNA?
PCR: Polymerase Chain Reaction
Also known as “Molecular photocopying”
Invented by Kary Mullis - mid 1980s
The real story behind PCR ….
Taq polymerase: Thermus aquaticus
3 steps: Denature, Anneal & Elongate
PCR protocol - an overview
Theory and
Application
of PCR
PCR
Animation follows
* 92 - 94oC for 30 - 60 sec (denature)
* 37 - 72oC for 30 - 60 sec (anneal)
* 72oC for 30 - 60 sec (elongate) (60 sec per kb target sequence length)
* 25 - 35 cycles only (otherwise enzyme decay causes artifacts)
* 72oC for 5 min at end (to allow complete elongation of all product DNA)
End of Human Genome lecture #
Suggested Reading:
Books
1. Genomes by TA Brown
2. Molecular Cell Biology by Lodish et al
3. Molecular and Cell Biology - Schaum series
Research Papers
4. Trends Biotechnol 1994: 12, 280-86
5. Trends Biochem 2001: 26, 268-270