Physical Mapping - Bioinformatics - Lecture Slides, Slides of Bioinformatics

Download Physical Mapping - Bioinformatics - Lecture Slides and more Slides Bioinformatics in PDF only on Docsity! Physical Mapping I Docsity.com 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 Docsity.com 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 Docsity.com 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 Docsity.com 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 Docsity.com 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 Docsity.com 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 Docsity.com 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 Docsity.com 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 Docsity.com Fig 4.12 Partial Digest a REI site a L b b atl RET site atb+e RE site _ 4 Partial digestion with a restriction endonuchease G) Source DNA al ad a al a b+etd cra c+d a Docsity.com Restriction Site Mapping • Problems – Uncertain lengths • Gel electrophoresis gives error up to 5% – Fragments may be too short to measure – May lose fragments Docsity.com