Genomics and 

Proteomics
Contents
Bacterial artificial Chromosomes
• Accommodate a large
insert of DNA~150Kb,
based on the F plasmid.
• Circular, supercoiled
BACs resist breakage
• Fig 24.8 Map of the BAC
vector,pBAC108L.
Cloning sites: HindIII and
BamHI; Selection tool:
CmR; the origin of
replication: oriS; The genes
governing partition of
plasmids to daughter cells:
ParA and ParB)
Two Strategies used in Human Genome
Project

• The Clone-by-Clone Strategy

RFLPs, VNTRs, STSs (including ESTs
and microsatellites) as markers
• Shotgun Sequencing
The Clone-by-Clone Strategy

• A Map-then-Sequence strategy
• The whole genome is mapped by finding markers
regularly spaced along each chromosome.
• Sequence the clones used in the mapping and then
place the sequences in order so they can pieced
together.
2. Sequence-Tagged Sites (STS)
• Short sequences,
about 60-1000bp
long, that can be
detected by PCR.
• Fig 24.9 Sequence-
tagged sites. Red: PCR
primers; Green: a large
cloned piece of DNA;
Blue: PCR products as
STSs.
Microsatellites (one kind of STSs)
• STSs are useful in physical mapping or locating specific
sequences in the genome ， but they are worthless as
markers in traditional genetic mapping unless they are
polymorphic.
• Microsatellites (random repeat sequences with core usually
only 2-4bp long) are ideal as markers for both linkage and
physical mapping. They are highly polymorphic and also
widespread and relatively uniformly distributed in the human
genome.
• Scientists designed PCR primers that flank the repeats at
each locus and got these sequences as mapping markers.
ESTs (expressed sequence tags)
• These are STSs generated from cDNA libraries.
• Represent genes that are expressed in the cell from which the
mRNAs were isolated.
• Avoid most introns
Fig 24.10 Mapping with STSs
Radiation Hybrid Mapping
• BACs are so small relative to a whole human
chromosome that creating a BAC contig of a whole
chromosome would be unbearably laborious.
• A method to find linkage between STSs that are even
farther apart than those that could fit into a singe BAC.
• Use ionizing radiation to break human chromosomes
into pieces and doom human cells with hamsters cells to
form hybrid cells that contain only some of the human
chromosome fragments.
• To detect which STSs will be detected in the hybrid
cells. The more often they are together, the closer
together they are likely to be on a human chromosome.
Shotgun Sequencing Strategy
• assemble a BAC library of the
human genome.
• Sequence both ends of the DNA
insert of each clone (about 500bps
long, named STC) .
• Pick a seed BAC, subclone the DNA
into plasmid vectors and sequence
them.
• Assemble the sequence and find
overlapping BACs from STC.
• BAC walking: Sequence BACs with
minimal overlap at each end and
repeat.
• Fig 24.11 Shotgun-sequencing
method.
• But in fact, they sequence the inserts
at random to get about 35 billionnt of
sequence and then use computer
program to find areas of overlap
among the sequences and piece them
together
.
Sequencing Standards
• Working draft: 90% complete, error rate
of up to 1%.
• Final draft: error rate of less than 1/10000
have as few gaps as possible.
Working draft and finished version of the human
genome

• The working draft shows that the genome contains

only about 25,000-40,000 genes.
• About half of the genome has derived from the
action of tranposons.
• Dozens of human genes appear to have come via
horizontal transmission from bacteria.
• Total size of the genome is about 3.2GB
• Finished version was more complete and more
accurate. (Fig 24.14)
24.3 Application of Genomics

• Structural genomics: Finding out the sequences of

genomes.
• Applications of structural genomics:
1. Functional genomics: probing the pattern of gene
expression in a given cell type at a given time
2. Positional cloning: Finding genes involved in
genetic traits, especially genetic diseases.
Functional Genomics Techniques

• DNA Microarrays and Microchips

• Serial Analysis of Gene Expression (SAGE)
• Deletion analysis
• Location target sites for transcription factors
• Genome-wide expression analysis
DNA Microarrays and Microchips
• DNA microarray: different DNAs are spotted on one chip
and covalently attached by ultraviolet radiation to a thin
silane layer on top of the glass. (Fig 24.16)
• DNA microchip: probe is oligonucleotides synthesized right
on the surface of a chip. (Fig 24.17 )
If we need some n-mer probes, We can generate 4n
different such probes in only 4n cycles.
A 16mer oligonucleotides are calculated to be unique in
the human genome. So we can use minimum 64 cycles to
get a microchip which can check all the human genome
in one reaction.
• eg1. Use DNA microarray technique to examine the effect
of serum on the RNAs made by a human cell.
• Fig 24.18 Using a DNA chip. Fluorescent cDNA hybridized with the probes
in the chip. cDNAs from serum stimulated cells are labeled red whereas cDNAs
from serum starved cells were labeled green. Spots 2&4: Genes more active in the
presence of serum. Spots 3: Genes more active in the absence of serum. Spot 1:
Gene equal active in these two conditions.