CONSTRUCTION OF HUMAN GENE MAP

THROUGH MAP INTEGRATION- FROM

GENETIC MAP TO PHYSICAL MAP TO

SEQUENCE MAP

Preety Sweta Hembrom

M.Sc Genomic Science

MAP INTEGRATION

Mapping is identifying relationships between

genes on chromosomes

Two broad categories of map:

1. Genetic map

2. Physical map

GENETIC MAP

Describes the order of genes or other markers and the spacing between them on each chromosome.

Use of genetic markers.

DNA based marker can also serve as markers.

Value of genetic map is that an inherited disease can be located on the map.

Used to find the exact location of several important disease genes.

DNA MARKER

1. RFLPS (RESTRICTION FRAGMENT LENGTH

POLYMORPHISMS):

Defined by the presence or absence of a

specific site- restriction site.

If 2 related but different DNA molecules are

cut with the same restriction enzymes, a

segment of different lengths are produced.

And RFLP is the difference between two

DNA sequences that affect a restriction site.

2. SIMPLE SEQUENCE LENGTH

POLYMORPHISMS (SSLPS)

Arrays of repeat sequences that display length variations, different alleles containing different numbers of repeat units.

Two types of SSLP:

I. Minisatellites:-

Also known as variable number of tandem repeats (VNTRs)

Defined by the presence of a nucleotide sequence that is repeated several times.

II. MICROSATELLITES

simple tandem repeats (STRs)

Whose repeats are shorter, usually dinucleotide or

tetra nucleotide units.

Polymorphic because the number of repeats may

vary.

Scored by determining their length by PCR.

Fragments are separated by electrophoresis.

Primers is labeled with fluorescent dye.

Human genome contains 5870 markers.

3. SNPS (SINGLE NUCLEOTIDE

POLYMORPHISMS)

Positions in a genome where some

individuals have one nucleotide and others

have a different nucleotide.

Some of which also give rise to RFLPs.

In the human genome there are at least

1.42 million SNPs, only 100 000 of which

result in an RFLP.

LOD SCORE METHOD FOR ESTIMATING

RECOMBINATION FREQUENCY

Imperfect pedigrees are analyzed statistically, using

a measure called the lod score (Morton, 1955).

This stands for logarithm of the odds that the genes

are linked and is used primarily to determine if the

two markers being studied lie on the same

chromosome.

If the LOD analysis establishes linkage then it can

also provide a measure of the most likely

recombination frequency.

THE LOD SCORE

Computerized LOD score analysis is a simple way to

analyze complex family pedigrees in order to determine

the linkage between a trait and a marker, or two

markers.

The method briefly, works as follows:

Establish a pedigree

Make a number of estimates of recombination frequency

Calculate a LOD score for each estimate

The estimate with the highest LOD score will be

considered the best estimate.

LOD SCORE

The LOD score is calculated as follows:

LOD = Z =Log10 probability of birth sequence with a given linkage

probability of birth sequence with no linkage

By convention, a LOD score greater than 3.0 is considered

evidence for linkage.

On the other hand, a LOD score less than -2.0 is considered

evidence to exclude linkage.

GENETIC MAP AS A FRAMEWORK FOR

PHYSICAL MAP CONSTRUCTION:

PHYSICAL MAP

Determination of physical distance between

two points on chromosome. Distance in base pairs

Example: between physical marker and a

gene.

Need overlapping fragments of DNA Requires vectors that accommodate large inserts

Examples: cosmids, YACs, and BACs

CONTD.

Divided into 2 groups:

Low Resolution Physical mapping:

i. Cytogenetic map

ii. cDNA map

iii. Contig map

High Resolution Physical mapping:

i. Macrorestriction map

ii. RH mapping

iii. Sequence map

LOW RESOLUTION PHYSICAL MAPPING

1. CYTOGENETIC MAP

Chromosomal mapping.

Genes or other identifiable DNA fragments

are assigned to their respective

chromosome.

Based on the distinctive banding patterns.

Used to locate genetic markers.

2. CDNA MAP

Shows the position of expressed DNA regions.

Synthesized in the laboratory using mRNA as a template.

Can be mapped to genomic regions.

Provide the chromosomal location of the genes whose functions are currently unknown.

3. CONTIG MAPS

Bottom up mapping.

Involves cutting the chromosome into small pieces.

Can be verified by FISH which localizes cosmids to

specific regions within chromosomal bands.

Consist of a linked library of small overlapping

clones.

FLUORESCENT IN SITU HYBRIDIZATION (FISH)

FISH is an optical mapping.

FISH enables the position of a marker on a chromosome or extended DNA molecule to be directly visualized.

In optical mapping the marker is a restriction site and it is visualized as a gap in an extended DNA fiber.

In FISH, the marker is a DNA sequence that is visualized by hybridization with a fluorescent probe.

FLUORESCENT IN SITU HYBRIDIZATION

HIGH RESOLUTION PHYSICAL MAPPING

1. MACRORESTRICTION MAPS

Single chromosome is cut into large pieces.

Depicts the order of and distance between

sites at which rare- cutter enzymes cleave.

Simplest way to construct is to compare the

fragment sizes.

The scale of restriction mapping limited by

the sizes of the restriction fragments.

2. RADIATION HYBRID MAPPING

Shows an estimated distance between genetic

markers.

A scientist exposes DNA to measure doses of

radiation.

Useful for ordering markers in regions where highly

polymorphic genetic markers are scarce.

Bridge between linkage map and sequence maps.

3. SEQUENCE MAPPING

Sequence tagged site (STS) mapping.

Short sequence of DNA.

Exact location and order of the bases of

sequence must be known.

May occur only once in the chromosome.

COMMON SOURCES OF STS

I. EXPRESSED SEQUENCE TAGS(ESTS)

Obtained by analysis of cDNA clones.

cDNA is prepared by converting mRNA into

double stranded DNA.

Thought to represent the sequences of the

genes being expressed.

II. SIMPLE SEQUENCE LENGTH

POLYMORPHISMS(SSLPS)

• Most genomes contain repeats of three or four nucleotides

• Length of repeat varies due to slippage in replication

• Use PCR with primers external to the repeat region

• On gel, see difference in length of amplified fragment

NEED TO INTEGRATE PHYSICAL AND GENETIC

MAPS:

STS based mapping has its limitations.

DNA fragments may lost or mistakenly mapped to a

wrong position.

DNA fragment may become contaminated with host

genetic material.

Comparing and integrating STS based physical

maps with genetic, RH, cytogenic maps.

CONTD

Ultimate objective of human genome was to

complete DNA sequence for the organism.

In order to locate genes and other interesting

features.

So in order to master sequence of chromosome

involves several sequencing method:

1. Sequence assembly by clone Contig method.

2. Whole genome Shotgun sequencing.

1. SEQUENCE ASSEMBLY BY THE CLONE

CONTIG METHOD:

Conventional method for obtaining sequence of a

eukaryotic genome.

Genomes are broken into fragments of upto 1.5 Mb

in length.

Built up by identifying clones containing overlapping

fragments.

2. WHOLE GENOME SHOTGUN SEQUENCING

Uses a map to aid assembly of the master

sequence

Used to speed up the acquisition of contig

sequence data for large genomes such as human

genome.

At least two libraries are used.

MAPPING PHASE OF THE HUMAN GENOME

PROJECT

Discovery of RFLPs.

In 1987 first human RFLP map was published.

Goal was a genetic map with density of one marker

per 1 Mb.

The 1994 map contained 5800 markers of which

over 4000 were SSLPs.

SEQUENCING THE HUMAN GENOME

The whole genome shotgun was first proposed as

an alternative to contig method.

The first draft sequence of an entire human

chromosome (22) was published in December

1991.

Finally on June 26, 2000 Francis Collins and Craig

Venter jointly announced the completion of project.

HUMAN GENOME

Contains about 20,000 to 30,000 genes.

Only 1-2% is coding region.

Rest are “junk DNA”.

Some sections of the human genome have a

sequence almost exactly the same as equivalent

sections in other vertebrates .

FUTURE OF THE HUMAN GENOME PROJECT

Completion of a finished sequence is not only the

goal.

Use of comparative genomics.

Direct development of new drugs and therapies

against cancer and other diseases.