Chapter 22 - Genomics and DNA Analysis Notes

Genomics Overview

  • Genome: Total genetic composition of an organism.
  • Genomics: Molecular analysis of the entire genome of a species.
  • Phases of Genome Analysis:
    • Mapping: Determining locations of genes.
    • Sequencing: Determining the exact sequence of nucleotides.
  • Functional Genomics: Studies gene interactions and their contribution to traits.
  • Proteomics: Study of proteins encoded by the genome and their interactions.

Key Historical Advances in DNA Sequencing

  • 1973: First DNA sequencing.
  • 1976: Walter Fiers sequences the 3,569 bp bacteriophage MS2 genome.
  • 1982: Frederick Sanger sequences the 48,502 bp bacteriophage lambda genome (14x MS2).
  • 1995: Craig Venter sequences the 1,830,137 bp Haemophilus influenzae genome (38x lambda).
  • 1996: André Goffeau leads team to sequence the 12.1 million bp Saccharomyces cerevisiae genome (7x Haemophilus).
  • 1997: International Consortium sequences the 4.6 million bp Escherichia coli genome (2.5x Haemophilus).
  • 2001: Human genome (3.1 billion bp) sequenced by International Consortium led by Francis Collins and Celera (Craig Venter).

Chromosome Mapping Techniques

1. Cytogenetic Mapping
  • Also called cytological mapping.
  • Uses microscopy to map genes relative to chromosomal band locations.
2. Linkage Mapping
  • Uses genetic crosses to map genes relative to their linked locations.
  • Distances measured in map units (centiMorgans).
3. Physical Mapping
  • Utilizes DNA cloning/sequencing to measure distances in base pairs between genes.

Comparisons and Gene Linkage

  • Correlation between gene linkage and base pairs varies across chromosomes.
  • Example organisms with mapping unit per base pairs:
    • Saccharomyces cerevisiae: 2,700,000 bp/unit
    • Drosophila melanogaster: 1,000,000 bp/unit
    • Mus musculus: 2,000,000 bp/unit
    • Homo sapiens: 1,200,000 bp/unit

Cytogenetic Mapping Details

  • Distinguishing Chromosomes: Based on size, centromeric location, and banding patterns.
  • Relies on specific dyes for banding patterns, mapped to identify gene locations.
  • Accuracy is about 5 million bp resolution.
In Situ Hybridization
  • Allows for localization of genes on chromosomes using DNA probes (FISH).
  • Fluorescent probes are used to visualize gene locations.

Linkage Mapping via Molecular Markers

  • DNA segments at specific locations can serve as genetic markers.
  • Markers can vary among individuals (polymorphic).
  • Microsatellites: Short repetitive sequences useful in genetic mapping.
    • Common microsatellite: $(CA)^n$ (where n ranges).
    • Amplified with PCR to create sequence-tagged sites (STS).
    • Heterozygotes display two distinct bands on a gel.

Chromosome Walking and Primer Walking

  • Physical mapping: Involves cloning many pieces of chromosomal DNA and organizing them as contigs (overlapping regions).
  • Walking techniques are employed to find specific genes of interest.

Overview of Genome Sequencing

Types of Vectors for Cloning
  1. Yeast Artificial Chromosomes (YACs): Accept large inserts but yield low DNA amounts.
  2. Bacterial Artificial Chromosomes (BACs): Medium inserts with high DNA yield.
  3. P1 Artificial Chromosomes (PACs): Similar to BACs.
  4. Cosmids: Used for sequencing genomes, can hold shorter sequences but allow for higher DNA yields.
Sequencing Methods
  • Shotgun Sequencing: Fragments are sequenced as a group, does not require extensive mapping.
  • High-throughput sequencing: Parallel sequencing of multiple DNA fragments across genomes.
  • Tools: Technologies like Single-molecule real-time sequencing and others with varying read lengths.

Human Genome Project Goals

  • Initiated on October 1, 1990, with goals including:
    1. Genetic linkage map of human genome.
    2. Physical map of human genome.
    3. Complete DNA sequence of human genome.
    4. Development of technologies for genome information management.