Homology-based cloning

Molecular Genetics

Homology-based cloning

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Often an investigator wants to isolate, from a particular organism, a gene orthologous to one already isolated from another organism.
Other investigators may be interested in isolating from the same organism other members of a gene family (paralogues) for which at least one cloned member is available.
The homologous genes (orthologues or paralogues) may be used as probes to isolate the target genes from a library.

Homology-based cloning may be effective when the protein whose gene it is wished to clone is a known member of a multi-gene family. Often, amino acid sequence alignments of family members reveal a particularly conserved sequence. Degenerate oligonucleotides that encode that sequence can be made and used in library screening. Top
Homology-based cloning may be effective when the gene of interest has been isolated from another organism. If the genes in these two organisms have not diverged too far since their evolutionary separation, nucleic acid probes made from the already isolated gene may be able to hybridize specifically with the target gene. Thus, if the genes from several organisms are available, that from the most closely related should be used. When gene sequences are available from several species, the screening specificity may be enhanceable by determining which regions of the sequence are most conserved. Probes made from those regions have a greater likelihood of hybridizing at reasonable stringencies. Top
When sequences from two separated parts of the gene are moderately conserved, degenerate oligonucleotides based on the two sequences can be used to attempt to amplify the intervening sequence by PCR. Top
An amplified RT-PCR product or a degenerate oligonucleotide may be used in nucleic acid hybridization to screen the colonies or plaques of a cDNA library for clones containing complementary sequences. Top
Positive clones obtained from an ordinary or an expression cDNA library can be used as nucleic acid hybridization probes to screen a library of genomic DNA to identify clones containing the gene that can produce the corresponding mRNA. Top
Positively reacting genomic library clones need to be further examined to :
- Identify where on the clone the gene is located.
- Prove that the clone actually contains the gene.

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E-mail inquiries to U. Melcher------------Last Updated: 14 October, 1999