Genetic Markers

1
Genetic Markers

• Lecture 2
• Strachan and Read Chapter 13

2
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

3
RFLPs
4
Microsatellite repeats
5
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

6
Affymetrix chips
From Affymetrix website
7
SNP genotyping on DNA chips
8
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

9
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.

10
Finding genes in the candidate region

• In the old days, or if your organisms 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!

11
Genes between the markers D4S10 and D4S181 on
chromosome 4
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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