Genomics

Mapping Genomes
Maps of genomes can be divided into 2 types
-Genetic maps
-Abstract maps that place the relative
location of genes on chromosomes
based on recombination frequency
-Physical maps
-Use landmarks within DNA
sequences, ranging from restriction sites
to the actual DNA sequence
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 Physical Maps
Distances between “landmarks” are measured
in base-pairs
-1000 basepairs (bp) = 1 kilobase (kb)
Knowledge of DNA sequence is not necessary
There are three main types of physical maps
-Restriction maps
-Cytological maps
-Radiation hybrid maps
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Physical Mapping of DNA
 Background

Given a sequence of DNA, how do we figure

out where on some larger chromosome
the sequence lies?

?
 Background II
Look for markers that match in both the
chromosome and the shorter sequence.
– Markers: Usually short, precisely defined sequences

match!
 Background III
How do we create the original map?

• Generate fingerprints (markers) with:

– Restriction site mapping
– Hybridization
 Background IV
But why? Can’t we just expand the
sequence assembly techniques we’ve
already learned?
NO! (with one exception)
Why not?
A chromosome isn’t just 150k bps long.
… Human chromosomes range in length
from 51 million to 245 million base pairs.
 Restriction Site Mapping
In this situation, the fingerprint is the
length between restriction sites of
given enzymes (recall from previous
lectures).
• Make three copies of target DNA: strings A, B, C.
• Apply one enzyme (α) to string A, another (β) to string B, and
both (α and β) to string C.
• Line up the fragments in A and B so they match C: this is the
double
6 digest problem.
14 7 5
8 3 15 4
6 2 3 9 7 1 4
 Restriction Site Mapping II
A variant is the partial digest approach:
• Use only one enzyme, but allow it to act
for different time periods. Different
restriction sites will be recognized.
6 14 7 5

Fragment sites: 6, 20, 27, 32; 14, 21, 26; 7, 12; and 5
 Restriction Site Mapping III

6
20 6

… 6 14

14
21 14
14 7

6 etc
14 … 7 5

Fragment sites: 6, 20, 27, 32; 14, 21, 26; 7, 12; and 5
 Hybridization Mapping
• Check whether specific small sequences
(called probes) bind (hybridize) to
fragments (clones)
• The fingerprint is the subset of probes that
successfully hybridize to the clone.
• If some portion of one clone’s fingerprint
matches another, they are likely to be from
overlapping regions of the target.
 Physical Maps
Restriction maps
-The first physical maps
-Based on distances between restriction
sites
-Overlap between smaller segments can be
used to assemble them into a contig
-Continuous segment of the genome

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 Physical Maps
Cytological maps
-Employ stains that generate reproducible
patterns of bands on the chromosomes
-Divide chromosomes into subregions
-Provide a map of the whole genome, but
at low resolution
-Cloned DNA is correlated with map using
fluorescent in situ hybridization (FISH)
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Physical Maps

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 Physical Maps
Radiation hybrid maps
-Use radiation to fragment chromosomes
randomly
-Fragments are then recovered by fusing
irradiated cell to another cell
-Usually a rodent cell
-Fragments can be identified based on
banding patterns or FISH
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 Physical Maps
Sequence-tagged sites
-An STS is a small stretch of DNA that is
unique in the genome
-Only 200-500 bp
-Boundary is defined by PCR primers
-Identified using any DNA as a
template
-STSs essentially provide a scaffold for
assembling genome sequences 21
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 Genetic Maps
Genetic maps are measured in centimorgans
-1 cM = 1% recombination frequency
Linkage mapping can be done without
knowing the DNA sequence of a gene
-Limitations:
1. Genetic distance does not directly
correspond to actual physical distance
2. Not all genes have obvious
phenotypes 24
 Genetic Maps
Most common markers are short repeat
sequences called, short tandem repeats,
or STR loci
-Differ in repeat length between individuals
-13 form the basis of modern DNA
fingerprinting developed by the FBI
-Cataloged in the CODIS database to
identify criminal offenders
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 Genetic Maps
Genetic and physical maps can be correlated
-Any cloned gene can be placed within the
genome and can also be mapped genetically

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 Genetic Maps
All of these different kinds of maps are stored
in databases
-The National Center for Biotechnology
Information (NCBI) serves as the US
repository for these data and more
-Similar databases exist in Europe and
Japan

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 Whole Genome Sequencing
The ultimate physical map is the base-pair
sequence of the entire genome

-Requires use of
high-throughout
automated
sequencing and
computer analysis

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 Whole Genome Sequencing
Sequencers provide accurate sequences for
DNA segments up to 800 bp long
-To reduce errors, 5-10 copies of a genome
are sequenced and compared
Vectors use to clone large pieces of DNA:
-Yeast artificial chromosomes (YACs)
-Bacterial artificial chromosomes (BACs)
-Human artificial chromosomes (HACs)
-Are circular, at present 29
 Whole Genome Sequencing
Clone-by-clone sequencing
-Overlapping regions between BAC clones
are identified by restriction mapping or STS
analysis
Shotgun sequencing
-DNA is randomly cut into smaller fragments,
cloned and then sequenced
-Computers put together the overlaps
-Sequence is not tied to other information 30
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 The Human Genome Project
Originated in 1990 by the International Human
Genome Sequencing Consortium
Craig Venter formed a private company, and
entered the “race” in May, 1998
In 2001, both groups published a draft
sequence
-Contained numerous gaps

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 The Human Genome Project
In 2004, the “finished” sequence was published
as the reference sequence (REF-SEQ) in
databases
-3.2 gigabasepairs
-1 Gb = 1 billion basepairs
-Contains a 400-fold reduction in gaps
-99% of euchromatic sequence
-Error rate = 1 per 100,000 bases
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 Characterizing Genomes
The Human Genome Project found fewer
genes than expected
-Initial estimate was 100,000 genes
-Number now appears to be about 25,000!
In general, eukaryotic genomes are larger and
have more genes than those of prokaryotes
-However, the complexity of an organism is
not necessarily related to its gene number
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Characterizing Genomes

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 Finding Genes
Genes are identified by open reading frames
-An ORF begins with a start codon and
contains no stop codon for a distance long
enough to encode a protein

Sequence annotation
-The addition of information, such as ORFs,
to the basic sequence information
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 Finding Genes
BLAST
-A search algorithm used to search NCBI
databases for homologous sequences
-Permits researchers to infer functions for
isolated molecular clones

Bioinformatics
-Use of computer programs to search for
genes, and to assemble and compare
genomes 38
 Genome Organization
Genomes consist of two main regions

-Coding DNA
-Contains genes than encode proteins

-Noncoding DNA
-Regions that do not encode proteins

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 Coding DNA in Eukaryotes
Four different classes are found:
-Single-copy genes : Includes most genes
-Segmental duplications : Blocks of genes
copied from one chromosome to another
-Multigene families : Groups of related but
distinctly different genes
-Tandem clusters : Identical copies of
genes occurring together in clusters
-Also include rRNA genes 40
 Noncoding DNA in Eukaryotes
Each cell in our bodies has about 6 feet of
DNA stuffed into it
-However, less than one inch is devoted to
genes!

Six major types of noncoding human DNA

have been described

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 Expressed Sequence Tags
ESTs can identify genes that are expressed
-They are generated by sequencing the
ends of randomly selected cDNAs
ESTs have identified 87,000 cDNAs in
different human tissues
-But how can 25,000 human genes encode
three to four times as many proteins?
-Alternative splicing yields different
proteins with different functions 42
Alternative Splicing

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Variation in the Human Genome
Single-nucleotide polymorphisms (SNPs)
are sites where individuals differ by only one
nucleotide
-Must be found in at least 1% of population
Haplotypes are regions of the chromosome
that are not exchanged by recombination
-Tendency for genes not to be randomized
is called linkage disequilibrium
-Can be used to map genes 44
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