Markers, mapping, and linkage (i.e. Where's the gene?)

Goal: To find a locus, relative to markers that are closely linked. The first step is high resolution mapping. Fortunately, in the case of humans, the genome is entirely sequenced.

NCBI - the human genome

Example: A segment of Xq27

Markers and landmarks

Genes

STSs - a common language

ESTs

RFLPs

Human SNP database

(Common SNP rate 1 per 1kbp, however SNP deserts exist)

Reference reading: The birth and death of human SNPs

"A dinucleotide repeat such as (dC-dA)n . (dG-dT)n was reported to occur in the human genome as many as 50,000 times with n varying from 10 to 60. This type of reiterated sequence has been termed a Simple Sequence Repeat (SSR) (Jacob et al., 1991), microsatellite (Litt and Luty, 1989) or Short Tandem Repeat (SSR) Edwards et al. (1991). " source: http://www.nal.usda.gov/pgdic/Probe/v2n1/simple.html

SSRs - Example

(rate 1 per 10 kbp)

DNA Forensics Problem Set

VNTR tutorial

Gene mapping using isolated chromosomes and sub-chromosome regions.

Somatic cell hybrids

Monochromosomal Somatic Cell Hybrid Panel - cheap!

GeneBridge4 Radiation Hybrid Panel

Conservation of synteny

In situ hybridization - a distant view

Autoradiography manual

Getting the image - liquid emulsion

http://www.umanitoba.ca/faculties/agricultural_and_food_sciences/plant_science/COURSES/CYTO/l13/l13.html

FISH pictures - Univ. of Wisconsin

Human cytogenetic landmarks

Dendrimere probes from Genisphere

The "Starfish" method

Chromosome painting

http://www.digitalscientific.co.uk/images/7colfish_image_a.jpg

Digital Scientific - Fluorescence imaging

Zooming in:

Long-range restriction maps using Pulse-Field Gel Electrophoresis

20 kbp-200 kbp (low end window) -> 1.4-9.0 Mbp (upper end)

application of CpG-rich enzymes, such as

Not I GCGGCCGC

Sac II CCGCGG

BssHII GCGCGC

A suspicious pattern - bands all of the same size with these enzymes, spanning islands.

Grabbing the region:

YACs, PACs, BACs - applied to the generation of large contigs

Chromosome walking - creating an ordered library (human vs. non-human approach)

Tests for clone chimerism, using somatic cell library end fragments.

Testing for STS's and progress

DNA Sequencing, if it is not already done, and perhaps at the same time, look for the genes by

Exon trapping

CpG island mapping

Transcript mapping

Predictive programs - where are the introns? - where are the coding regions, based on codon usage?

Linkage

Genetic Linkage

Detecting and measuring linkage in humans

The LOD Score

Physical maps and positional cloning

Reference: CHROMOSOMAL MAPPING TOOLS

Reference: PHYSICAL MAPS AND POSITIONAL CLONING