Gene walking and chromosome jumping
Gene walking is basic method of genetic engineering that allows identification of unknown regions flanking a known DNA sequence. Thus gene walker does not have a clone of a gene but the known sequence. It is synonymously used for chromosome or genome walking. There are several walking strategies which are based on restriction, digestion, ligation and polymerase chain reaction (PCR). The four of them are described here:
Inverse PCR: Inverse PCR method is one of the variations of PCR and involves the amplification of DNA with only one known sequence. This means that PCR is carried out by using one sequence from which primers can be obtained. It involves the series of restriction, digestion and ligation resulting in the formation of circularized or looped fragment. The only limitation of this method is that the reaction requires large amount of DNA (2-10μg) and dependency on available restriction sites.
Vectorette PCR: Vectorette is DNA fragment consisting mismatched bases which offer internal binding site for a sequencing primer. In this method, sequencing primer is used to obtain approximately 2Kb of sequence from a single strand of PCR fragment. Thus PCR is carried out with specific and Vectorette primer to yield an array of fragments of about 500bp for the sequencing. This method is used for sequencing orthologous gene regions such as exons and introns.
Universal fast walking: Universal fast walking allows fast, direct and economical determination of flanking sequence of the genome. It does not require restriction, digestion or ligation and hence the availability of restriction sites in the flanking region. The method is very important for the sequencing of unclonable and unsequenceable DNA segments.
Targeted walking: The method is modification of PCR where in two sets of primers are used in two successive reactions. The first primer set is a primer specific target sequence and another set is nonspecific walker primer. This combination of primers carries out the amplification of unknown DNA sequences adjacent to short stretch of known sequence.
Importance of gene walking methods: The most advantageous is that no need for construction of DNA libraries. Otherwise, construction, screening of genome libraries or identification of unknown regions of the genome from the libraries is very time consuming and laborious task. The amplification requires very little information about the sequences or probes subjected for screening.
Chromosome jumping is also one of the basic tools of molecular biology which involves physical mapping of genome. Jumping the chromosome indicates rapid movement along the chromosome in search of particular gene of interest. Thus it is used to bypass regions difficult to clone and cannot be mapped by gene walking. But walking and jumping processes are correlated. For jumping on the chromosome, the DNA of interest is cut into fragments with restriction enzymes and ligated to form circularized loop. A primer designed from a known sequence is used to sequence the circularized loop. This primer is used to jump 100kb or more intervals and additional jumps till it reaches the known sequence on the loop. In this process, the sequences not reachable by gene walking are sequenced; walking and jumping can be performed alternatively to sequence the whole chromosome.
Importance of chromosome jumping: Jumping of the chromosome is useful method for the cloning of genes responsible for human genetic disorders like cystic fibrosis. In this gene of interest is located directly on the map and cloned. It is also useful for identification; isolation and cloning of the gene whose products are unknown or cannot be determined. Jumping on chromosome has also been useful to get molecular marker and the gene of interest in close vicinity to each other so that cloning of very large fragment is avoided.
Advancement in PCR, their versions and variations are abundant but gene walking and chromosome jumping approaches are still the methods of choice for molecular biologists because of their speed, simplicity and versatility.
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