DNA variation
Seems a boring topic, but useful for things like
- DNA fingerprinting
- Finding disease genes like CF, HD and lots of others
Human genetic variation
- find about 1 difference every 1000bp
- means 3,000,000 differences between any two people
- if look at a third person, find differences between that person and the other two
- so lots of variation between people
- differences in DNA sequences between people are DNA polymorphisms
o if a particular DNA polymorphism is useful for mapping studies (such as finding a genetic disease gene) it is called a DNA marker
Four different classes of DNA polymorphisms
- SNPs
o Single base-pair differences
o Most abundant
o May or may not have any effect on a gene, depending on where it is located
§ If in coding region could be a missense or nonsense (or could be silent mutation)
§ If outside of coding region could effect splicing or transcription
§ If outside coding region could have no effect at all
o For ones that we think have no effect, then there is no natural selection acting on those alleles
§ Indicates a common ancestor for any two people who share a SNP
§ Because all humans share so many SNPs, indicates that all humans have a common ancestor who lived not too long ago……
- Microsatellites
o One, two, or three base sequence repeated in tandem 15-100 times
o Probably arise from errors in DNA replication, like shown in Fig. 11.3
o Found in about 200,000 different places in human genome
o Highly polymorphic – many different alleles in human population
- Insertions/Deletions (Indels)
o Inserting or deleting DNA that is NOT in a micro- or minisatellite
o Easily found by gel electrophoresis
- Minisatellites
o Repeats of 20-100bp long sequences
o Repeated many times (up to thousands)
o Highly polymorphic
o Often find same repeat or very similar repeat in several different places in genome
DNA fingerprinting
- Alec Jeffreys found that minisatellites are highly polymorphic and most occur at different sites in genome
- Developed original DNA fingerprinting procedure
o Isolate DNA
o Cut DNA with restriction endonucleases that cut just outside of the minisatellite
o Run on agarose gel
o Denature DNA and blot onto nylon membrane (Southern blot)
o Fix DNA on membrane
o Add labeled probe – probe is minisatellite sequence
o Hybridize
o Wash off unbound probe
o Detect (autoradiography or fluorescent)
o See something like Fig. 11.15
- Now use STRs (a class of microsatellites), do PCR, and run on gel to detect
Haplotype mapping for positional cloning
- can find genes using SNPs and doing haplotype mapping
- haplotypes are formed by sequential mutations in a small genomic region (Fig. 11.25)
- since formation of a SNP is a rare event, it is very unlikely that two individuals will share several SNPs on the same chromosome by chance. Rather, it probably means that they are descended from the same ancestor
- to map genes by haplotype mapping, you first get a bunch of people, some of whom have the phenotype and some who are unaffected
o We assume that everyone who has the disease has a mutation that arose long ago, and that that ancestor had a particular haplotype in that region of the chromosome
- over time, the kind of thing in Fig. 11.26 happens as recombination occurs during meiosis
- now you look at groups of SNPs and see if any of them are always associated with your phenotype of interest (like the 58bp microsatellite was associated with HD)
- the resolving power of this technique is great because there are so many SNPs scattered throughout the genome