physical mapping of the genome

1. http://www.free-powerpoint-templates-design.com
Topic: Physical Mapping of the
Genome.
Gene Analysis &
Genomics.
Presented By: Minahil Khalid.
Presented to: Dr. Humaira Yasmeen.

2. Content
What is physical mapping.
Assembly of physical mapping.
Restriction Enzyme Fingerprinting.
Marker Sequences .
Hybridization Assay .
Physical Mapping without cloning.
Conclusion.

3. Physical Mapping.

4. It is a map of genetic markers made by analyzing a
genomic DNA sequence directly.
It tracks the chromosomal region where specific
genes are found.

5. Assembly of Physical
Mapping.

6. After a genome has been fragmented and
the fragments cloned to generate a genomic
library, it is necessary to assemble the cloned
fragments in the same order as found in
chromosome from which they derived.

7. Two or more clones that can overlap makeup a contig.
Physical markers are used to identify the overlaps and
get mapped on the genome thereby creating a physical
map.
A physical map of chromosome is exactly the same in
that different kinds of markers are identified and mapped
along the chromosome.

8. Three general methods for mapping have been
developed:
• Restriction Enzyme Fingerprinting.
• Marker Sequences.
• Hybridization Assays.

9. Restriction Enzyme
fingerprinting.

10. The principle of Restriction Enzyme
Fingerprinting was originally
developed for the nematode
Caeno-rhabditis elegans and yeast.

11. Procedure:
• Creation of genomic library in the
cosmids.
• Digestion of the cosmids clone using
Hind III RE leaving staggered ends.
• Labelling of the fragments using dATP
in the presence of reverse
transcriptase
• HindIII was destroyed by heating.
• Double digestion of fragments by
Sau3A.
• Fragments were separated on high
resolution gel.
• Detection by autoradiography

12. Except for microbial genomes
restriction enzyme fingerprinting
applied to the genomes such as
Drosophila, Arabidopsis, and human
genome .

13. The reasons for this were two-folds:
• First, cosmids with an insert size of 40kb relatively
have few sites for SRE.
• Secondly , the problems of detecting overlaps from
fingerprint patters grow exponentially as size of
genome being mapped increases.

14. Two developments have lead to re-emergence in restriction
enzyme fingerprinting.
1. Development of high throughput method of fingerprinting using
large insert clones e:g BAC and PAC clones .

15. Agarose Mapping gel showing
human PACs digested with HindIII.
Clone are present in triplicate:
• Verify stability during propagation.
• Check possibility of cross
contamination.

16. 2.Development of restriction fragment mapping was the creation
powerful software called fingerprinted contigs(FPC) .
Benefits of FPC Software:
• Use to compare the fingerprints of different clones and determine which
one overlaps.
• The largest contig constructed consist of 9534 clones .

17. Marker Sequences.

18. “Sequences that have physical location on
chromosome”.
1. Sequence Tagged sites(STSs).
2. Sequence Tagged connectors(STCc).

19. It is a short region of DNA About 200-300 bases long
whose exact sequence is found nowhere else in genome.
STSs
A better method to develop STS markers is to create a
chromosome specified library in phage M13 .

20. STCc
They are long STSs and were first
proposed by Venter to sequence the
human genome .

21. SNPs.
These are single base pair positions
in genomic DNA at which different
sequence alternatives(allele) exist in
a population .

22. 1. 3 possible SNP variants for T/A
base pair
2.A single base change in the β-
globin gene that destroys a restriction
site for endonuclease MstII
generating a restriction fragment
length polymorphism (RFLP)
• Amino acid sequence changes
from glutamate to valine cause
the sickle cell disease.

23. Problem with the PCR in the
detection of STS and SNPs.
1. The method is labour intensive.
2. Cost of synthesizing large number
of different primers.
3. STSs are defined by the PCR
conditions that generate it.

24. AFLP.
It is a diagnostic fingerprinting
technique that detects genomic
restriction fragments in that they
resembles the RFLP technique.

25. • Isolation and digestion of genomic DNA
simultaneously using EcoRI and MseI .
• The former has a 6 bp recognition site and the
latter a 4 bp recognition site.
• DNA fragments will amplify and separated on
denaturing polyacrylamide gel.
• Heat inactivation of the RE ligated the genomic
DNA fragments to EcoRI and MseI adapters.
• DNA fingerprint detected by autoradiography.
Procedure:

26. Hybridization Assays.

27. Chromosomal walking.
It is a technique used to clone a gene
from its known markers and is used in
modifications in cloning and
sequencing .

28. • It is desired to clone DNA sequences of gene
B for which no probe is available and
identified genetically .
• Fragment 1 contains sequence of gene A
• Fragment 1 clone use as a probe to identify
overlapping sequence of 2,3,4,5 and so on.
Possible to walk along the chromosome until
gene B is reached.

29. Hybridization
Mapping.

30. Five kinds of probes representing known
repetitive sequences(centromeric,telomeric,
17S and 5S ribosomal and long terminal
repeat) are hybridized and use to identify the
clones containing the unique DNA .

31. • Hybridization identifies overlapping
clones
a)Clones for use as probes are
randomly picked (*) from a given set of
cosmids whose map order is not known
b)Gaps in the map caused by the lack of
probes

32. Physical Mapping
without cloning.

33. It is a technique for constructing ordered, genome-
wide, high-resolution restriction maps from single,
stained molecules of DNA, called "optical maps"
1.Optical Mapping.

34. 3 major STEPS:
1) Gelation.
2) Addition of restriction
enzyme & Mg Ions
3) Fluorescence Microscopy

35. 2. Radiation Hybridization mapping:
• Developed for constructing long-range maps of mammalian chromosomes.
• It determines not only the distances between deoxyribonucleic acid (DNA)
markers but also their order on the chromosomes.

37. Conclusion:
Each of the mapping methods
described above has its advantages
and disadvantages and no one
method is ideal.

38. Thank You
Any Question?