Structural Genomics Key Concepts

Structural Genomics

• Genetic and physical maps are used to determine the order of genes on a chromosome and their approximate distance apart.
• DNA sequence determination is performed using dideoxynucleotides that halt replication at a specific base.
• DNA fragments that differ by a single base can be separated using polyacrylamide gels.
• Sequencing reactions generate a few hundred bases of sequence.
• Whole genomes can be sequenced by cloning random small DNA genomic fragments, sequencing them, and then reassembling the genome sequence based on the overlap between the sequenced fragments.
• Massive computing power is required to assemble the sequenced fragments and determine the locations of genes within the genome.

Restriction Mapping

• Analysis of total genomic DNA and allocation or identification of restriction sites on genome using restriction enzymes.
• Restriction enzymes are used to digest the genome into fragments that can be processed for automated DNA sequencing.
• The resulting fragments can be fractionated on agarose gels to determine the restriction sites and gene orientation.
• Examples of restriction enzymes used include EcoRI and BamHI.

DNA Sequencing

• The process of determining the order of the nucleotides (A, T, G, C) in a genome.
• Types of DNA sequencing methods include:
  • Dideoxynucleotide method
  • Automated Sequencing
  • Maxam and Gilbert method (Chemical cleavage)
  • Sanger method (Enzymatic or Chain termination method)

Automated Sequencing

• The process uses dideoxynucleotides labeled with different acceptor dyes to terminate DNA synthesis.
• Sequencing reactions can be performed in a single tube and the products separated on a gel or using a capillary tube.
• The intensity and wavelength of the fluorescent emission are measured as the DNA fragments move past a laser Argon beam and fluorescence detector.
• This information is fed directly into a computer, assigning and storing the resulting sequence.

Maxam and Gilbert method (Chemical cleavage)

• A chemical method for cleaving the sugar–phosphate backbone of a radio-labelled DNA fragment at specific bases.
• Specific chemicals are used to modify individual DNA bases or sets of bases prior to cleavage of the sugar–phosphate backbone with piperidine at the modified bases.
• The separation of the cleaved products using high-resolution polyacrylamide gel electrophoresis allows assignment of individual bases within a DNA sequence.

Sanger method (Enzymatic or Chain termination method)

• An alternative sequencing approach based upon the faithful replication of DNA using a DNA polymerase.
• Dideoxynucleotides are incorporated into a newly replicated DNA chain to generate DNA fragments that ended at a specific base.
• The sequencing reaction is split into four separate parts, each with a mixture of the four nucleotide triphosphates and a single dideoxynucleotide triphosphate.