Lecture 2 Strachan and Read Chapter 13 Genetic Markers Lecture 2 Strachan and Read Chapter 13

Polymorphism in human DNA Millions of sites in human DNA are different between individuals Single nucleotide polymorphisms (SNPs) in genes or in non-coding DNA may or may not affect phenotype SNPs can cause Restriction fragment length polymorphisms (RFLPs) if in a restriction enzyme site Tandem repeat sequences (or microsatellies), such as dinucleotides (CA)n, tri- and tetra-nucleotides, that are variable for the number of repeats. Most polymorphisms are in non-coding DNA – there is more of it, and mutations are not selected against

RFLPs

Microsatellite repeats

Rapid genotyping using chips To do serious amounts of genotyping, need something quicker than the last 2 methods Affymetrix and Illumina DNA microarrays (chips) with up to 106 probes corresponding to both alleles of SNPs across genome Label test DNA and hybridise to chip Scan chip and read out which allele for each SNP is hybridised (both if heterozygous) Enables rapid genome-wide genotyping

Affymetrix chips From Affymetrix website

SNP genotyping on DNA chips

The major stages in carrying out the project Collect as many affected families as possible. Assess all individuals clinically, take blood samples for DNA. Genome scan: Genotype families with 400-500 markers evenly distributed over the whole genome, using PCR based methods and automated processing, if possible. Analyse results for linkage to determine location of gene - the "candidate region". Identify genes in candidate region by database searching. Compare sequence of candidate genes in patients and controls, to identify disease-specific mutations

Genome scans Genome Scan is genotyping a collection of families with the genetic disease using hundreds of genetic markers from all over the genome. “Brute force" approach is necessary because of the great size of the human genome (3000 megabases or 3x109bp). Using hundreds of markers ensures unknown gene will be close enough to one or two of them to show genetic linkage. The aim is to find linkage with two markers, one of which is on each side of the disease gene. Then you would know that the disease gene must be in the candidate region of the genome between the two markers, a few million bases of DNA.

Finding genes in the candidate region In the old days, or if your organism’s genome has not been sequenced, you had to do a lot of DNA cloning and analysis in the lab Now you just look in the database!

Genes between the markers D4S10 and D4S181 on chromosome 4

What next? Screen genes in candidate region to identify the correct one (next lecture) Use it to perform presymptomatic diagnosis by DNA testing, to detect gene carriers or pregnancies at risk Understand more of the biology of the disease (e.g. cystic fibrosis gene codes for a chloride ion channel) Design new drug therapies - and possibly gene therapy