Types of Genome Map

• 1) Genetic linkage map

• 2) Physical Map

1
 Genetic linkage Map
• Depicts the order of genes and relative distance between the genes.

• Maps were made by tracing the inheritance of multiple traits.

• (eg) Hair colour, Eye colour etc through several generations.

• Gnetic linkage map possible because of normal biological process : crossing

over which occurs during meiosis.

• (eg) Brown eye + brown hair x Blue eye + Blond hair

Crossing over in sperm cell: Brown eye + Blond hair

2
 PHYSICAL MAPS: Low resolution physical Mapping
• CHROMOSOMAL MAP

• Genes or DNA fragments : assigned to their respective chromosomes.

• Distances are measured in base pairs.

• Markers can be associated with respective bands.(FISH).

• Traits observable in whole organism.

• Best chromosomal map : used to locate fragments of about 10 kb.

• Improvements in FISH allow 2 to 5 kb.

• Modification in insitu hybridisation : using chromosomes at stage in cell division where

they are less compact : increases map resolution.

3
 Low resolution Physical Map
• CDNA MAP

• Shows positions of expressed DNA region relative to particular chromosomal regions or

bands.

• cDNA is synthesised using mRNA as a template.

• cDNA can be mapped to genomic regions.

• cDNA’s help identify parts of the genome with most biological and medical
significance.

4
 Metaphase chromosomes
• Mataphase chromosome: Important for analysis of cancer cytogenetics.

• Malignant cells (solid tumours or leukemia) are grown in short term culture.

• Generate metaphase chromosomes.

• Stain with Giemsa or quinacrine

• Shows loss of chromosomal segments or translocations.

5
Typical Metaphase spread

6
 FISH
Principle
Hybridisation of labelled probe (DNA or RNA) with specially prepared chromosomes.

Probes: Initially radiolabelled.

Tagged with floorescent markers.1kb in size

Chromosomes are held on slides pretreated to expose and denatureDNA.

Chromosomes are loaded with labelled probe.

Incubated under anneling conditions, washed to remove unhybridised probe.

Location of the hybridised probe : determined by fluorescence microscopy.

FISH has played a mojor role in correct ordering of YAC.

7
Fluorescence In Situ Hybridisation

8
Sample FISH

9
 Interphase FISH

• Positive for 9,22 chromosomal rearrangement.

10
21 trisomy

11
 Cancer
• Acute myelocytic leukemia. Chromosome 8.

12
 Translocation of 18 and 3
• Green probe: chromosome 3
• Red probe: chromosome 18

13
 Use of FISH
• Determining the number of copies of a chromosome (e.g. chromosome
21).

• Determining the presence of polyploid cells in leukemias, lymphomas

and solid tumors

• Identifying translocations (a translocation is an exchange of a region

of one chromosome with that of another chromosome)

• Identifying large (usually greater than megabase) deletions

• Gene mapping

14
 Uses of FISH

• Locus specific probes: Bind to paricular region in a chromosome.

• Alpoid or centromeric repeat probes: Generated from repeat sequences

found in the middle of each chromosome.

• Whole chromosome probe: small probes which bind to different

sequences along the length of a chromosome. Helps in identifying
chromosomal abnormalities. Is called the spectral karyotype.

15
 Fluorescent probes for FISH
• The basic elements of FISH are DNA probe and target sequence.

Before hybridization, the DNA probe is labeled by various means, such as nick
translation, random primed labeling, and PCR.

• Two labeling strategies are commonly used: indirect labeling and direct labeling .

• For indirect labeling, probes are labeled with modified nucleotides that contain a hapten.

• Direct labeling uses nucleotides that have been directly modified to contain a
fluorophore.

• The labeled probe and the target DNA are denatured.

• Combining the denatured probe and target allows the annealing of complementary DNA
sequences.

• If the probe has been labeled indirectly, an extra step is required for visualization of the
nonfluorescent hapten that uses an enzymatic or immunological detection system. .

16
• FISH is faster with directly labeled probes.

• Indirect labeling offers the advantage of signal amplification by using several

layers of antibodies.

• This might therefore produce a signal that is brighter compared with

background levels

17
FISH

18
 High resolution physical mapping
• Top down mapping : (macro restriction map)

• Single chromosome cut into large pieces , (cut by enzymes)ordered and subdivided.

• Analysis by pulse field gel electrophoresis.

• Existing genetic map : use probes to serve as anchor points on the physical map.

• Maps with more continuity and smaller gaps.

• Resolution low , not helpful in finding specific genes.

• DNA pieces in regions measuring about 100,000 bp to 1 Mb

19
 High resolution physical mapping
• Bottom – up mapping (contig maps)

• Preparation of completely ordered genomic library.

• Start with individual clones that are fingerprinted.

• Arranged with overlapping of DNA segments in clones.(contigs)

• Clone size from 10,000 bp to 1Mb.

• Advantages of contig maps: linked library of overlapping clones representing complete

chromosomal segment.
• Useful for finding genes in small area.(under 2 Mb).

• Disadvantage :All regions are not clonable as difficult to extend over large stretches of
chromosome.

20
 Chromosome banding
• Treatment of chromosomes to reveal characterestic patterns of horizontal
bands.

• Resembles a barcode.

• Banding lends each chromosome a distinctive appearance.

• Helps in identification of 22 pairs of human autosomes and the X and Y

chromosomes without ambiguity.

• It also permits the recognition of chromosome deletions, chromosome

duplications and other types of structural rearrangements of chromosomes.

21
Banding in chromosomes

22
 Banding in chromosomes
• G-banding : trypsin digestion and then Giemsa stain
• light and dark bands
• dark region AT rich

• R-banding : Reverse banding

• dark regions GC rich

• C-banding : stains centromeres

• Q-banding : fluorescent pattern, quinacrine stain.(similar to g-banding)

• T-banding : visualise telomeres.

•

23
 Genome Variation
• Differences in the sequence of DNA from one person to the other.

• Variations are found throughout the genome on everyone of the 46

chromosomes.

• Parts of genomes: hotspots of variability : hundreds of possible variations of a

sequence.

• Parts of genome : very stable : Don’t vary much between individuals.

• Majority of variations are found outside of genes in the junk DNA : does not
affect a persons characterestics : Hence mutations not harmful and variations
can occur without causing harm.

24
• Genes in contrast tend to be stable.

• Since mutations that occur in genes are often harmful to an individual.

Why study variations ?

• Has practical applications

• 1) Genome mapping
• 2) Screening for genetic diseases
• 3) Forensic technologies like DNA fingerprinting

25
 Single nucleotide Plymorphism :SNP’s
• Important variation

• Risk factor for disease

• (eg) SNP’s in Azheimers: Gene called apolipoprotein E or Apo E.

• Apo E has 2 SNP’s and hence 3 alleles.

• Alleles: E2, E3, E4.

• Alleles differ from each other by 1 DNA base.

• The corresponding proteins differ from each other by 1 aminoacid.

26
 Copy number variation(CNV’s)
• Human genome comprise roughly around 30000 genes.

• Normal dosage : 2 copies.

• Recent discoveries have revealed that large segments of DNA, ranging in size
from thousands to millions of DNA bases, can vary in copy-number.

• This can lead to dosage imbalance

• Differences in DNA sequence in individuals: uniqueness

• CNV’S comprise contains 3 times the total nucleotide content of SNP’s

27
 How would CNV mapping help?
• A global CNV map will transform medical research in four areas.

• 1) Hunting for genes underlying common diseases.

• 2) CNV map is being used to study familial genetic conditions.

• 3) There are thousands of severe developmental defects caused by

chromosomal rearrangements.

• 4)The CNV map is being used to exclude variation found in unaffected

individuals, helping researchers to target the region that might be involved.

The data generated will also contribute to a more accurate and complete
human genome reference.

28
 Research revealed…….
• 12% of the human genome was copy number variable in the 270 DNA
samples tested.

• About 2900 genes, or 10% of those known, are encompassed by these CNVs.

• Approximately 2000 CNVs have been described.

• It is estimated that there could be thousands more CNVs in the human

population.

29
 CNV’s and diseases

• Most CNVs are benign variants that will not directly cause disease.

• There are several instances where CNVs that affect critical developmental
genes do cause disease.

• Recent reviews have listed 17 conditions of the nervous system alone –

including Parkinson’s Disease and Alzheimer’s Disease – that can result from
copy number variation.

30
 Types of genes that are copy number variable !!!
• Genes that are involved in the immune system and in brain development and
activity – two functions that have evolved rapidly in humans – tend to be
enriched in CNVs.

• Genes that play a role in early development and some genes involved in cell
division – both critical to fundamental biology – are spared.

31
 Yeast artificial chromosome (YAC)
• Vector to clone DNA fragments larger than 100 to 3000 kb.

• Useful for cloning large genes.

• Yeast cells contain 16 chromosomes that contain varying amount of DNA.

• YAC first designed and reported by David Burke.

• Contains centromere, telomere and origin of replication sites.

• Above 3 regions are spliced into DNA in proper location and orientation helping in
replication of the artificial chromosome along with its natural chromosome.

• Target Dna flanked by telomeric regions marking the ends and iterspersed with
centomere vital for replication.

• Start site already present.

32
 Yeast artificial chromosome
• ADVANTAGES

• Allows insersion of fragment size 1,000,000 bp

to

• A plasmid that allows only up to 10,000 bp.

33
 Cloning human genomic DNA into YAC
• Genomic DNA is partially digested by restriction enzyme EcoR1. This helps
in obataining large DNA fragments.

• YAC is digested by Eco R1 and BamH1

• These two elements recombine at the EcoR1 sites and are covalently linked by
DNA ligases.

• A recombinant YAC vector, a YAC with genomic DNA inserted is produced.

• This vector can be used to infect yeast cells and generate an unlimited number
of copies.

34
YAC (yeast artificial chromosome)

35