You do not have permission to edit this page, for the following reasons:

This IP address has been blocked from editing Wikipedia.
This does not affect your ability to read Wikipedia pages.
Most people who see this message have done nothing wrong. Some kinds of blocks restrict editing from specific service providers or telecom companies in response to recent abuse or vandalism, and can sometimes affect other users who are unrelated to that abuse. Review the information below for assistance if you do not believe that you have done anything wrong.

The IP address or range 44.211.0.0/16 has been blocked (disabled) by ‪Blablubbs‬ for the following reason(s):

The IP address that you are currently using has been blocked because it is believed to be a web host provider or colocation provider. To prevent abuse, web hosts and colocation providers may be blocked from editing Wikipedia.
You will not be able to edit Wikipedia using a web host or colocation provider because it hides your IP address, much like a proxy or VPN.
We recommend that you attempt to use another connection to edit. For example, if you use a proxy or VPN to connect to the internet, turn it off when editing Wikipedia. If you edit using a mobile connection, try using a Wi-Fi connection, and vice versa. If you are using a corporate internet connection, switch to a different Wi-Fi network. If you have a Wikipedia account, please log in.
If you do not have any other way to edit Wikipedia, you will need to request an IP block exemption.

How to appeal if you are confident that your connection does not use a colocation provider's IP address:
If you are confident that you are not using a web host, you may appeal this block by adding the following text on your talk page: ((unblock|reason=Caught by a colocation web host block but this host or IP is not a web host. My IP address is _______. Place any further information here. ~~~~)). You must fill in the blank with your IP address for this block to be investigated. Your IP address can be determined here. Alternatively, if you wish to keep your IP address private you can use the unblock ticket request system. There are several reasons you might be editing using the IP address of a web host or colocation provider (such as if you are using VPN software or a business network); please use this method of appeal only if you think your IP address is in fact not a web host or colocation provider.

Administrators: The IP block exemption user right should only be applied to allow users to edit using web host in exceptional circumstances, and requests should usually be directed to the functionaries team via email. If you intend to give the IPBE user right, a CheckUser needs to take a look at the account. This can be requested most easily at SPI Quick Checkuser Requests. Unblocking an IP or IP range with this template is highly discouraged without at least contacting the blocking administrator.

This block will expire on 10:53, 20 August 2024.

Even when blocked, you will usually still be able to edit your user talk page, as well as email administrators and other editors.

For information on how to proceed, please read the FAQ for blocked users and the guideline on block appeals. The guide to appealing blocks may also be helpful.

Other useful links: Blocking policy · Help:I have been blocked
Your IP address is in a range that has been blocked on all Wikimedia Foundation wikis. The block was made by ‪AntiCompositeNumber‬. The reason given is Open proxy/Webhost: Visit the FAQ if you are affected .
- Start of block: 13:55, 18 August 2022
- Expiry of block: 13:55, 18 April 2025
Your current IP address is 44.211.87.112. The blocked range is 44.211.0.0/16. Please include all above details in any queries you make. If you believe you were blocked by mistake, you can find additional information and instructions in the No open proxies global policy. Otherwise, to discuss the block please post a request for review on Meta-Wiki. You could also send an email to the stewards VRT queue at stewards@wikimedia.org including all above details.

You can view and copy the source of this page:

((short description|DNA sequencing method))
((Multiple issues|
((cleanup|date=December 2021|reason=improper linkage, and text that assumes prior knowledge))
((More citations needed|date=December 2008))
))

'''Primer walking''' is a technique used to clone a gene (e.g., disease gene) from its known closest markers (e.g., known gene). As a result, it is employed in cloning and sequencing efforts in plants, fungi, and mammals with minor alterations. This technique, also known as "directed sequencing," employs a series of Sanger sequencing reactions to either confirm the reference sequence of a known plasmid or PCR product based on the reference sequence (sequence confirmation service) or to discover the unknown sequence of a full plasmid or PCR product by designing primers to sequence overlapping sections (sequence discovery service).<ref>((Cite web|last=Company|first=Azenta Life Sciences|title=Primer Walking FAQ|url=https://web.genewiz.com/primer-walking-faq|access-date=2021-11-20|website=web.genewiz.com|language=en))</ref>

== 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>

== Primer walking versus shotgun sequencing ==
Primer walking is an example of [[directed sequencing]] because the primer is designed from a known region of DNA to guide the sequencing in a specific direction. In contrast to directed sequencing, shotgun sequencing of DNA is a more rapid sequencing strategy.<ref>((Cite web|title=ScienceDirect.com ((!)) Science, health and medical journals, full text articles and books.|url=https://www.sciencedirect.com/|access-date=2021-11-20|website=www.sciencedirect.com))</ref>

There is a technique from the "old time" of genome sequencing. The underlying method for sequencing is the Sanger chain termination method which can have read lengths between 100 and 1000 basepairs (depending on the instruments used). This means you have to break down longer DNA molecules, clone and subsequently sequence them. There are two methods possible.<ref>((Cite web|title=genetics - Why do we use DNA sequencing methods such as shotgun?|url=https://biology.stackexchange.com/questions/21251/why-do-we-use-dna-sequencing-methods-such-as-shotgun|access-date=2021-11-20|website=Biology Stack Exchange))</ref>

The first is called chromosome (or primer) walking and starts with sequencing the first piece. The next (contiguous) piece of the sequence is then sequenced using a primer which is complementary to the end of the first sequence read and so on. This technique doesn't require much assembling, but you need a lot of primers and it is relatively slow.<ref>((Cite web|title=genetics - Why do we use DNA sequencing methods such as shotgun?|url=https://biology.stackexchange.com/questions/21251/why-do-we-use-dna-sequencing-methods-such-as-shotgun|access-date=2021-11-20|website=Biology Stack Exchange))</ref>

To overcome this problem the shotgun sequencing method was developed. Here the DNA is broken into different pieces (not all broken at the same place), cloned and sequenced with primers specific for the vector used for cloning. This leads to overlapping sequences which then have to be assembled into one sequence on the computer. This method allows for the parallelization of the sequencing (you can prepare a lot of sequencing reactions at the same time and run them) which makes the process much faster and also avoids the need for sequence specific primers. The challenge is to organize sequences into their order, as overlaps are not as clear here. To resolve this problem, a first draft is made and then critical regions are resequenced using other techniques such as primer walking.<ref>((Cite web|title=genetics - Why do we use DNA sequencing methods such as shotgun?|url=https://biology.stackexchange.com/questions/21251/why-do-we-use-dna-sequencing-methods-such-as-shotgun|access-date=2021-11-20|website=Biology Stack Exchange))</ref>

==Process==
The overall process is as follows:
A [[primer (molecular biology)|primer]] that matches the beginning of the DNA to sequence is used to synthesize a short DNA strand adjacent to the unknown sequence, starting with the primer (see [[Polymerase chain reaction|PCR]]).
The new short DNA strand is sequenced using the chain termination method.
The end of the sequenced strand is used as a primer for the next part of the long DNA sequence, hence the term "walking".

The method can be used to sequence entire chromosomes (hence "chromosome walking").<ref>((Cite journal| doi = 10.1016/0378-1119(79)90097-0| volume = 5| issue = 2| pages = 111–126| last = Chinault| pmid = 376402| first = A. Craig|author2=John Carbon| title = Overlap hybridization screening: Isolation and characterization of overlapping DNA fragments surrounding the ''leu2'' gene on yeast chromosome III| journal = Gene| date = Feb 1979))</ref> Primer walking was also the basis for the development of [[shotgun sequencing]], which uses random primers instead of specifically chosen ones.

==See also==
* [[Chromosome jumping]]
* [[Chromosome landing]]
* [[Shotgun sequencing]] – an alternative method, using random, rather than consecutive, sub-strands.

==References==

((reflist))

[[Category:DNA]]
[[Category:Molecular biology]]

Pages transcluded onto the current version of this page (help):

Primer walking (edit)
Template:Ambox (view source) (template editor protected)
Template:Category handler (view source) (protected)
Template:Cite book (view source) (protected)
Template:Cite journal (view source) (protected)
Template:Cite web (view source) (protected)
Template:Cleanup (view source) (template editor protected)
Template:DMC (view source) (template editor protected)
Template:DMCA (view source) (template editor protected)
Template:Dated maintenance category (view source) (template editor protected)
Template:Dated maintenance category (articles) (view source) (template editor protected)
Template:FULLROOTPAGENAME (view source) (template editor protected)
Template:Find sources mainspace (view source) (template editor protected)
Template:Main other (view source) (protected)
Template:More citations needed (view source) (template editor protected)
Template:Multiple issues (view source) (template editor protected)
Template:Multiple issues/styles.css (view source) (template editor protected)
Template:Ns has subpages (view source) (protected)
Template:Pagetype (view source) (protected)
Template:Reflist (view source) (protected)
Template:Reflist/styles.css (view source) (protected)
Template:SDcat (view source) (protected)
Template:SUBJECTSPACE formatted (view source) (template editor protected)
Template:Short description (view source) (protected)
Template:Short description/lowercasecheck (view source) (protected)
Template:Terminate sentence (view source) (template editor protected)
Module:Arguments (view source) (protected)
Module:Category handler (view source) (protected)
Module:Category handler/blacklist (view source) (protected)
Module:Category handler/config (view source) (protected)
Module:Category handler/data (view source) (protected)
Module:Category handler/shared (view source) (protected)
Module:Check for unknown parameters (view source) (protected)
Module:Citation/CS1 (view source) (protected)
Module:Citation/CS1/COinS (view source) (protected)
Module:Citation/CS1/Configuration (view source) (protected)
Module:Citation/CS1/Date validation (view source) (protected)
Module:Citation/CS1/Identifiers (view source) (protected)
Module:Citation/CS1/Utilities (view source) (protected)
Module:Citation/CS1/Whitelist (view source) (protected)
Module:Citation/CS1/styles.css (view source) (protected)
Module:Disambiguation/templates (view source) (protected)
Module:Find sources (view source) (template editor protected)
Module:Find sources/config (view source) (template editor protected)
Module:Find sources/links (view source) (template editor protected)
Module:Find sources/templates/Find sources mainspace (view source) (template editor protected)
Module:Message box (view source) (protected)
Module:Message box/ambox.css (view source) (protected)
Module:Message box/configuration (view source) (protected)
Module:Namespace detect/config (view source) (protected)
Module:Namespace detect/data (view source) (protected)
Module:Ns has subpages (view source) (protected)
Module:Pagetype (view source) (protected)
Module:Pagetype/config (view source) (protected)
Module:Pagetype/disambiguation (view source) (protected)
Module:Pagetype/setindex (view source) (protected)
Module:Pagetype/softredirect (view source) (protected)
Module:SDcat (view source) (protected)
Module:String (view source) (protected)
Module:Text (view source) (template editor protected)
Module:Unsubst (view source) (protected)
Module:Wikitext Parsing (view source) (protected)
Module:Yesno (view source) (protected)

Return to Primer walking.

Retrieved from "https://en.wikipedia.org/wiki/Primer_walking"