The Human Genome And Gene Mapping

Readings

Jorde, Carey, Bamshad & White: Medical Genetics, 3rd edition, C.V. Mosby Publishing, 2005.

Structure of the Human Genome Chapter 2, 17-22
Detection of variation at the DNA level Chapter 3, 43-55
Gene Mapping and cloning Chapter 8, 160-189

Objectives

The student will:

Understand the different types of genetic variation.
Be able to distinguish between genetic mapping and physical mapping.
Understand the basic concept of genetic linkage analysis
Understand different gene mapping techniques .

The Human Genome Project

Goal is/was determination of the complete sequence of the human genome
- genes which define our phenotype
- genes whose alteration leads to disease
Characterization of single gene disorders, multigene disorders
3 x 109 bp (<5% encodes genes)
24 different chromosomes
First draft of the sequence of human genome published in 2000

Mapping

Genome maps are detailed constructs of the order and/or position of genetic markers and DNA sequence.
Maps are of two types:
- Genetic maps - use recombination frequencies of markers at meiosis
- Physical maps - identify exact location of DNA sequence in the genome

Human Genome Mapping

Individual chromosomes isolated, and used to prepare genomic DNA libraries in yeast artificial chromosomes
Unusual world-wide collaboration
New technology of robotic production lines (PCR tests)
Information shared on web (900 printed pages)

Genetic Markers

Freely recombine during meiosis unless they are physically close together
Linked markers usually inherited as a set

Genetic Maps

Genetic maps dependent on linkage analysis and recombination frequency:

Units of scale
- Human = centimorgan (cM)
- 1 cM = recombination fraction of 1/100 meioses
- Equivalent to 0.7 - 1 Mb of DNA

Definitive genetic map

Assembled by Genethon laboratory (Paris)
Used microsatellite markers

Map Your Disease

Find a large family with a clearly defined disease
Using PCR check with 400 primers pairs, at about 11 cM intervals
Automation
Markers used in mapping
Restriction fragment polymorphisms (RFLP)
Minisatellites
Microsatellites

Results

At least one marker should co-segregate with disease
Go to Human Genome Project to find linked loci
Fill in the gaps
Scan the human genome database for sequences associated with active genes in your marker

Proving You Have the Gene

Transgenic mouse - prove gene has biological function related to disease
Knockout mouse - inactivate gene and show disease develops

Linkage Calculation

LOD score = log of the odds score = statistical estimate of whether two loci are likely to lie near each other on a chromosome
If they are they are likely to be inherited together
LOD score > 3 generally taken to indicate that the two loci are close (1 to 1,000)
Determine recombination frequencies - can range from 0% (no recombination) to 50% (crossing over in half of meioses)
50% recombination frequency would be observed from two markers on different chromosomes
Not calculated by hand...

Probabilities

Probability found by multiplying together the probabilities of each event happening independently
For instance: I need to be at work by 8 AM. For this to happen I have to set my alarm clock, I have to be lucky with traffic, and I have to find a parking space
Probability (work at 8) = Probability (set alarm) x Probability (no traffic) x Probability (parking space)

Physical Maps

Plot the actual location of DNA sequences on chromosomes
Units of scale = base pairs
Clone maps
Radiation hybrid maps - radiation breaks chromosomes
Fluorescent in situ hybridization (FISH)
Karyotype changes

Useful Terms

Genetic markers - provide the landmarks to plot positions within the genomic landscape
May be associated with phenotypic characteristics such as inherited diseases
Many not associated yet with observable phenotype = DNA sequence polymorphisms

Hapmap (Haplotype Map)

CEPH Donors

Reconsent from living donors
Local IRB gave permission to use samples from deceased donors
No names or other identifying information
No medical information
Only genotype information and sex released on Internet
More samples collected than used

Array Technology

Possible because of new technology
Allows better resolution than comparative genomic hybridization (CGH) on chromosomes

Array Analysis

Uses include gene expression, CGH, SNP analysis, protein analysis
Differences lie in material on array (DNA, cDNA, oligonucleotides, antibodies, proteins)
Choice of targets on array
Sensitivity - minimum region
Mosaics may not be detected
DNA - can do multiple tests in one experiment
Can compare multiple different individuals at multiple different targets