Homology-Based Cloning

Based on known cinnamate 4-hydroxylase sequences from Jerusalem artichoke and mung bean a homology search in an EST library identified an EST clone with 84-86% sequence identity, which was then used as a probe to isolate the CYP73A5 from a genomic library.  

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Kapitonov D, Bieberich E, Yu RK (1999) Combinatorial PCR approach to homology-based cloning cloning and expression of mouse and human GM3-synthase. Glycoconj J 16 337-350... 

Fig. 4 Homology based cloning. Conserved regions of known enzyme classes are used to design oligonucleotide primers, which are used to isolate the desired gene from plant cDNA...

Homology-based cloning is limited to enzymes with a known conserved sequence. If no consensus... 

The answer to this question is not known. However, homology-based molecular cloning techniques have led to the identification of literally hundreds of these receptors each of which conforms to the seven transmembrane domain pattern. The function of many of these receptors and their ligands are unknown such receptors are referred to as orphan receptors. Similarly, there is a considerable diversity of G-proteins. In particular, over 30 different a-subunit types have been described. 

We searched for isoprenyl diphosphate synthase gene sequences in P. abies using a homology-based approach. First, RNA was isolated from bark and wood of methyl jasmonate-treated spruce saplings from a single clone. This was then used as a template for reverse transcriptase PCR carried out with degenerate primers... 

Homology screening. Using oligonucleotide probes based on known receptor sequences, search cDNA libraries for homologous sequences which may code for related receptors. The clones are then isolated and sequenced and used in expression studies to confirm the identity of the receptor. 

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