MolCellEng_Notes5 - 1 Physical Mapping 1 Based on distances...

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1. Physical Mapping 1. Based on distances in base pairs between specific sequences found on the chromosome 2. Example: determination of physical distance in base pairs between physical marker and gene on chromosome 3. Needs overlapping fragments of DNA 4. Requires vectors that accommodate large inserts (cosmids, YACs, BACs) 5. Physical Markers 1. DNA sequences that vary between two related genomes 1. Referred to as DNA polymorphism (polymorphism = occur in different forms) 2. Usually not in a gene 3. Examples: RFLP, SSLP, SNP 6. RFLP = Restriction-fragment length polymorphism 1. Two sets of DNA, difference in bases near restriction sites. 2. Cut genomic DNA from 2 individuals with restriction enzyme 3. Run southern blot 4. Probe w/ diff pieces of DNA 5. Sequence difference creates different band pattern 7. SSLP = Simple-sequence length polymorphism … aka microsatellites 1. Genomes contain repeats of 3 – 4 nucleotides; length of repeat varies 1
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2. Use PCR with primers external to the repeat region [PCR obtains the repeating sequence out from the DNA sequence] 3. On gel, see difference in length of amplified fragment 4. smaller repeat region will move along gel and travel farther (b/c lighter) 8. SNP = Single-nucleotide polymorphism 1. One nucleotide difference in sequence of two organisms found by sequencing 2. Between any two humans, on avg. one SNP every 1,000 base pairs 9. Large insert vectors 1. Lambda phage (insert size: 20 – 30 kb) 2. Cosmids (insert size: 35 – 45 kb) 3. BACs and PACs (Bacterial and P1 artificial chromosomes, respectively) (insert size: 100 – 300 kb) 4. YACs (yeast artificial chromosomes) (insert size: 200 – 1000 kb)
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MolCellEng_Notes5 - 1 Physical Mapping 1 Based on distances...

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