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Physical Mapping I

Physical Mapping I. CIS 667 February 26, 2004. Physical Mapping. A physical map of a piece of DNA tells us the location of certain markers A marker is a short sequence Given a sequence and a chromosome - try to find the place of the sequence. C. D. H. G. B. C. A. G.

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Physical Mapping I

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  1. Physical Mapping I CIS 667 February 26, 2004

  2. Physical Mapping • A physical map of a piece of DNA tells us the location of certain markers • A marker is a short sequence • Given a sequence and a chromosome - try to find the place of the sequence C D H G B C A G

  3. Physical Mapping • Generally used to resolve regions much larger than 1 Mb (e.g. whole chromosomes) • Map is created by fragmenting the DNA molecule using restriction enzymes and then looking for overlaps • The pieces are too big to sequence, so this is not the same problem as fragment assembly!

  4. An Example - Cystic Fibrosis • Cystic fibrosis is a fatal disease • 1 in 25 Caucasians carries a faulty cystic fibrosis gene • Children who inherit faulty genes from both parents become sick • Best hope for a cure starts with finding the responsible gene • In the mid 80s nothing was known about the CF gene so a search was started for it

  5. An Example - Genetic Mapping • Restriction fragment length polymorphism (RFLP) was used to construct a map of the human genome with one marker every 10 million nucleotides • RFLP based on variability of certain nucleotides between different people • These cause restriction fragments of different lengths to be produced

  6. An Example - Genetic Mapping • A statistical study of 21 families over three generations narrowed the search led to an area of length 1 million bp on chromosome 7 between 2 markers • The presence of two phenotypes together more often leads to the conclusion that the genes are physically close due to the way crossover works to reshuffle genomic material

  7. An Example - Physical Mapping • Now physical mapping was needed to more precisely locate the CF gene • The DNA molecule was broken into pieces 50 Kb long • Now the correct ordering of the pieces had to be obtained • They are cloned to obtain a large number of copies in a clone library • Now the clones must be ordered

  8. An Example - Physical Mapping • The idea is to describe each clone using a fingerprint to describe the clones • Can be thought of as “key words” for the clones • Overlapping clones should have similar fingerprints X Y Z Q

  9. An Example - Physical Mapping • Fingerprints can be • Sizes of restriction fragments of clones • List of probes hybridizing to the clones • The CF gene was close to RFLP DS78 marker • A probe for this RFLP was used to find a clone containing it • The clone was sequenced at the end and a new probe designed to move closer to the gene - (chromosome walking)

  10. Physical Mapping • Two generic ways of obtaining fingerprints are • Restriction site analysis • Hybridization • Restriction site analysis locates the restriction sites of an enzyme on the target DNA • Generally applicable with smaller DNAs • Viral and mitochondrial DNA

  11. Physical Mapping • In hybridization mapping we check whether certain small sequences bind to fragments • More widely used nowadays, especially for large scale physical mapping • Due to lack of information and errors, we may not be able to produce a single contiguous physical map • May have lack of coverage or chimeric clones

  12. Restriction Site Mapping • There are two techniques for measuring the length of fragments between restriction sites • Apply two different restriction enzymes to the target DNA • Each enzyme cuts at a different location • Apply enzyme A, apply enzyme B, and also apply both A and B together • Now we have a three sets of fragments of various lengths

  13. Restriction Site Mapping • The discovery of the original ordering of these fragments is called the double digest problem

  14. Partial Digest Problem • A variant of the double digest approach is partial digest • Use just one enzyme, but for varying amounts of time • Fragments of different lengths will be produced

  15. Restriction Site Mapping • Problems • Uncertain lengths • Gel electrophoresis gives error up to 5% • Fragments may be too short to measure • May lose fragments

  16. Hybridization Mapping • Done using markers called STS (Sequenced Tagged Sites) • Uses PCR techniques to identify unique sequences • Verify whether the clone allows some probes to bind (hybridize) • See if the clones have overlapping sets of binding probes

  17. Hybridization Mapping • Problems • False negative • False positive • Chimeric clones • Deletion • Repeats (not for STS)

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