DNA Sequencing

  Molecular cloning, the isolation and amplification of relatively short stretches of chromosomal DNA, is frequently followed by sequencing the nucleotides in these fragments. DNA sequencing provides a meaning to distinguish genes from surrounding chromosomal material, to determine their possible function from the decoded protein structure, and to find a nucleotide mutation which can lead to dysfunctional protein associated with a particular pathology. For example, a single change in the CF gene can disrupt its critical function in the membranes of multiple cell types, causing cystic fibrosis (covered in detail in Dr. Holmes9 clinic). Likewise, a single inherited base changes in the myosin heavy chain gene specifically expressed in cardiac myocytes can disrupt the ability of the resulting protein to function properly as a component of the contractile apparatus, resulting in familial hypertrophic cardiomyopathy (you will here more about this pathology in Dr. Seidman9s clinic).

  Most DNA sequencing is performed via an enzymatic reaction using DNA polymerase and specific DNA chain terminators, called dideoxyribonucleotides. This technique has proven particularly amenable to automation, which has been a breakthrough for large-scale sequencing projects. The sequence of the entire yeast genome has recently been completed, and sequencing of the human genome is well on its way. The challenge ahead is to interpret both structural and regulatory information buried in the vast stretches of available sequences, which also encode the rules governing the selection of genes for expression in different tissues, and pathologic aberrations of this control.