Editing Primer walking (section)

== Primer walking: a DNA sequencing method ==
Primer walking is a method to determine the sequence of DNA up to the 1.3–7.0 kb range whereas [[chromosome walking]] is used to produce the clones of already known sequences of the gene.<ref>{{Cite book|url=https://www.worldcat.org/oclc/53956473|title=DNA viruses : a practical approach|date=2000|publisher=New York|others=Alan Cann|isbn=0-585-48411-2|location=Oxford|oclc=53956473}}</ref>  Too long fragments cannot be sequenced in a single sequence read using the [[chain termination method]]. This method works by dividing the long sequence into several consecutive short ones. The DNA of interest may be a [[plasmid]] insert, a [[Polymerase chain reaction|PCR]] product or a fragment representing a gap when sequencing a genome. The term "primer walking" is used where the main aim is to sequence the genome. The term "chromosome walking" is used instead when the sequence is known but there is no clone of a gene. For example, the gene for a disease may be located near a specific marker such as an [[RFLP]] on the sequence.<ref>{{cite book|last=Cann|first=Alan|title=DNA Viruses: A Practical Approach|publisher=Oxford University Press|year=1999|isbn=978-0-19-963718-8}}</ref> Chromosome walking is a technique used to clone a gene (e.g., disease gene) from its known closest markers (e.g., known gene) and hence is used in moderate modifications in cloning and sequencing projects in plants, fungi, and animals. To put it another way, it's utilized to find, isolate, and clone a specific sequence existing near the gene to be mapped. Libraries of large fragments, mainly bacterial artificial chromosome libraries, are mostly used in genomic projects. To identify the desired colony and to select a particular clone the library is screened first with a desired probe. After screening, the clone is overlapped with the probe and overlapping fragments are mapped. These fragments are then used as a new probe (short DNA fragments obtained from the 3′ or 5′ ends of clones) to identify other clones. A library approximately consists of 96 clones and each clone contains a different insert. Probe one identifies λ1 and λ2 as it overlaps them . Probe two derived from λ2 clones is used to identify λ3, and so on. Orientation of the clones is determined by restriction mapping of the clones. Thus, new chromosomal regions present in the vicinity of a gene could be identified. Chromosome walking is time-consuming, and chromosome landing is the method of choice for gene identification. This method necessitates the discovery of a marker that is firmly related to the mutant locus.<ref>{{Cite book|date=2015|title=Modern Applications of Plant Biotechnology in Pharmaceutical Sciences|url=http://dx.doi.org/10.1016/c2014-0-02123-5|doi=10.1016/c2014-0-02123-5|isbn=9780128022214}}</ref>

The fragment is first sequenced as if it were a shorter fragment. Sequencing is performed from each end using either universal [[primer (molecular biology)|primers]] or specifically designed ones. This should identify the first 1000 or so bases. In order to completely sequence the region of interest, design and [[Chemical synthesis|synthesis]] of new primers (complementary to the final 20 bases of the known sequence) is necessary to obtain contiguous sequence information.<ref>{{cite journal|title=Sequence analysis of genes and genomes|url=http://www.nslc.wustl.edu/elgin/genomics/bio4342/1archives/2004/Sterky.pdf|journal=Journal of Biotechnology|volume=76|issue=1|date=2000|pages=1–31|first1=Fredrik |last1=Sterky |first2=Joakim |last2=Lundeberg |doi=10.1016/s0168-1656(99)00176-5|pmid=10784293}}</ref>