Biol223-lecture21

Chapter 8: Genomics Overview

  • Focus on the mapping and sequencing of genomes.

  • Exercises include: 8.5, 8.6, 8.10, 8.14, 8.19

Key Techniques in Genomics

  • Restriction Enzymes: Cut DNA at specific sequences.

  • Cloning Vectors: Used for transferring DNA fragments into host cells.

  • Constructing and Screening Libraries: Methods for isolating specific DNA fragments.

  • Southern and Northern Blot Techniques: Used for detecting specific DNA or RNA sequences.

  • DNA Sequencing: Determines the order of nucleotides in DNA.

  • Polymerase Chain Reaction (PCR): Amplifies DNA sequences.

  • Recombinant DNA Technology: Combines DNA from different sources.

Restriction Enzymes

Definition

  • Restriction endonucleases (RE) hydrolyze the phosphodiester bonds in double-stranded DNA.

  • Found mainly in bacteria for protection against viruses; named for their source organism.

Restriction Sites

  • Specific sequences where restriction enzymes cut DNA, often palindromic.

  • Recognition probability: (1/4)^n, where n is the number of base pairs in the target sequence.

Commonly Used Restriction Enzymes

  • Ava I: Anabaena variabilis, recognizes C*C/T C G A/G G

  • Bam HI: Bacillus amyloliquefaciens, targets G*G A T C C

  • Eco RI: Escherichia coli RY 13, cuts G*A A T T C (similar formats for other enzymes).

Types of Cleavage

  • Blunt Ends: Fragments with no overhangs.

  • Sticky Ends: Fragments with overhangs that can base-pair with complementary ends.

Cloning Vectors

Types of Vectors

  • Plasmids: Circular dsDNA, typically carrying antibiotic resistance genes and unique restriction sites.

  • Bacterial Artificial Chromosomes (BAC): Cloning large DNA fragments (up to 300 Kb).

  • YAC (Yeast Artificial Chromosomes): Linear vectors for cloning very large inserts (up to 2 million bp).

  • Shuttle Vectors: Carry cloned genes between different host organisms.

Components of Vectors

  • Origin of Replication: Starting point for DNA replication.

  • Selectable Markers: Genes for antibiotic resistance or nutrients.

  • Unique Restriction Sites: For inserting foreign DNA.

Construction of Libraries

Genomic Libraries

  • Definition: A genomic library contains clones representing every DNA sequence of a genome.

  • Construction: DNA is partially digested with restriction enzymes to produce overlapping fragments.

Chromosomal Libraries

  • Specific type of genomic library focusing on individual chromosomes for easier gene isolation.

cDNA Libraries

  • Contain DNA copies of mRNA, reflecting gene expression at a specific time. Constructed from purified mRNAs.

Screening Libraries

cDNA Expression Libraries

  • Can express the cDNA it contains, allowing identification of specific proteins via antibodies.

Hybridization Probes

  • Used to identify specific DNA sequences in genomic libraries. Probes can be synthesized based on known gene sequences.

Applications in Gene Cloning

  • Gene cloning can complement mutations in cell mutants, allowing identification of genes responsible for specific traits.