Important Note

Tufts ended funding for its Open Courseware initiative in 2014. We are now planning to retire this site on June 30, 2018. Content will be available for Tufts contributors after that date. If you have any questions about this please write to edtech@tufts.edu.

Tufts OpenCourseware
Search
Author: Janet M. Cowan, Ph.D.
Color Key
Important key words or phrases.
Important concepts or main ideas.

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
    Results

Proving You Have the Gene

  • Transgenic mouse - prove gene has biological function related to disease
  • Knockout mouse – inactivate gene and show disease develops
    Linkage, Prediction, Recombination

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

Summary

  • Human Genome Project resulted in explosion of new technology
  • Raised ethical questions (who owns the genome?)
  • Will enable faster identification of disease related genes
  • May enable personalized treatments