The document discusses DNA sequencing and cloning disease genes in the human genome. It describes two methods for cloning a disease gene: 1) cloning a gene of known function by first isolating and sequencing the associated protein to design a DNA probe to identify the gene in a cDNA library, with examples including phenylalanine hydroxylase for phenylketonuria. 2) positional cloning by mapping the disease locus to a chromosome region containing many DNA markers, then testing the markers for co-segregation with disease to narrow down the region containing the gene.

1. DNA Sequencing
and
Cloning Disease Genes in the Human
Genome
By Tajuddin Shaik

2. DNA Sequencing

6. Cloning a Disease Gene of Known
Function
• In general, the first step in cloning a gene of known function is
to isolate the protein. Protein isolation requires an assay for
the protein, such as an enzyme activity assay where a product can
be measured. In essence one needs to know the function of
the protein in order to purify it.
• 1. Once the protein is purified, small pieces of the protein
are sequenced, i.e. the amino acid order is determined. Protein
sequencing is a difficult technique and it will only be possible to
get partial protein sequence. Based on the protein sequence,
a synthetic oligonucleotide DNA probe is
generated using the genetic code. This probe is used to search
a cDNA library, i.e.
isolate a molecular clone containing the gene for the protein.
• 2. Examples of genes of known function that have been
cloned (associated disease): phenylalanine hydroxylase (phenylketo
nuria),

10. Cloning a Disease Gene

11. Application of Cloning Gene