Transcript Genetic markers

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