5. How the Genome is Studied
Maps and sequences
Locus: location of a gene in a chromosome. Two genes are assorted (or
segregated, i.e. are on the same chromosome) if an offspring has about
50% chance of inheriting both characteristics (deduced from the genes)
from the same parent.
Recombination: due to crossing-over (when cells divide) between
chromosomes. If genes are closed together, there is a small chance of
separation due to crossing-over.
Genetic linkage map: constructed from observed segregation percentage,
of certain characteristics. It shows the order and relative distance between
genes (108 bp).
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Maps and Sequences (cont.1)
Physical map: tells the location of certain marquers (precisely known
sequences) within105 - 106 base pair. Physical map construction is among
the studied bioinformatics problems.
Large-scale sequencing: done by breaking apart several copies of the
piece to be sequenced (20 kbp) and by sequencing the (small) fragments
directly.
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Specific Techniques
To obtain maps and sequences
Produces nearly data that have errors (so algorithms are to be extended to
handle errors.
Virus and bacteria (organisms most used in genetic research)
Virus consists of a protein cap (capsid) with DNA (or RNA) inside
- cells starts producing-coded proteins which promotes viral DNA
replication (new capsids break the cell membrane and attack other cells).
- or DNA gets inserted into the host genome until...
A bacterium (a single-cell organism having one chromosome, like
Escherichia Coli) can multiply by simple DNA replication in very short
period of time.
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Cutting and breaking DNA
Restriction enzyme: cut DNA at certain specific point (restriction site)
Example: EcoRI cuts DNA at GATTC
-between the G and the first A
-the two strands, as GAATTC is a palindrome because
GAATTC = GAATTC
Endo(exo)-nuclease: cuts DNA internally (externally)
Shotgun method breaks apart DNA molecules. Some fragments (issued
from a purified DNA) are filtered and selected for cloning. Then they are
sequenced (to determine the purified DNA's sequence) or they are
collected to construct a cloning library.
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Copying DNA (DNA Amplification)
One molecule is not enough for experiences.
DNA Cloning : by using nature itself, by inserting the initial piece in an
organism (called host or vector) and by leaving the organism multiply
itself. Afterwards, it is killed to produce DNA recombinant.
Typical hosts:
-Plasmid: piece of circular DNA which exists in bacteria. Limit of
insert size: 15 kbp.
-Phage (virus) : insert gets replicated when the virus infects a host
colony. The size limit is 25 kbp, more with cosmids (40kbp, entire phage
is replaced by the insert.
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Copying DNA (Con.1)
-YAC (Yeast Artificial Chromosome): artificial made chromosome
where the insert lookslike an additional chromosome to the yeast
replication mechanism (100-500kbp). BAC is for Bacterial Artificial
Chromosome.
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PCR
PCR(Polymerase Chain Reaction): DNA polymerase is an enzyme that
catalyses elongation of a single strand of DNA, provided there is
template DNA to which this single strand is attached. The (small) double
stranded DNA at the beginning is called a primer.
Two steps are repeated :
1. Separation into two single strands by heat of the original double
stranded ADN
2. Addition of primers and DNA polymerase action to produce a double
strand.
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Reading and measuring DNA
Gel electrophoresis: a technique which is based on separation of
molecules by their sizes by moving to a definite direction by action of an
electric field, with speed inversely proportional to their size.
Technique for reading: first obtain all fragments from the DNA
molecules that end with an A (resp. T, G, C), then put them into four four
different tubes, finally classify (by gel electrophoresis) the segments in
each tubes according their size.
Example: Given the DNA molecule GACTTAGATCAGGAAACT, the
fragments which terminate by T are :
GACT, GACTT, GACTTAGAT, GACTTAGATCAGGAAACT.
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