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 A B G C H D G C

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!

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

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

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

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

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

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)

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

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

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

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

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

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

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

Hybridization Mapping Problems  False negative  False positive  Chimeric clones  Deletion  Repeats (not for STS)