Vectorette PCR

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Vectorette PCR is a variation of polymerase chain reaction (PCR) designed in 1988. [1] The original PCR was created and also patented during the 1980s. [2] Vectorette PCR was first noted and described in an article in 1990 by John H. Riley and his team. [3] Since then, multiple variants of PCR have been created. Vectorette PCR focuses on amplifying a specific sequence obtained from an internal sequence that is originally known until the fragment end. [4] Multiple researches have taken this method as an opportunity to conduct experiments in order to uncover the potential uses that can be derived from Vectorette PCR. [1]

Contents

Introduction

Vectorette PCR is similar to PCR with the difference being that it is capable of obtaining the sequence desired for amplification from an already known primer site. [5] While PCR needs information of already known sequences at both ends, Vectorette PCR only requires previous knowledge of one. [1] This means that is able to apply the method of PCR which needs sequence information from both ends to fragments of DNA that contain the information of the sequence at only one end and not the other. [6] [7] In order to achieve this, there are specific steps that this method must first go through. These steps have been researched for the purpose of discovering the scientific uses of Vectorette PCR and how they can be applied. [1]

Steps

Vectorette PCR can develop a strategy to bring about PCR amplification that is unidirectional. [8] Vectorette PCR comprises three main steps. [1] The first step includes utilizing a restriction enzyme in order to accomplish digestion of the sample DNA. [1] [6] The DNA that is to be utilized for the purpose of investigation has to be capable of being digested by restriction enzymes that are appropriate for that gene otherwise the DNA fragments that form the general population cannot be created. [9] After that is completed, a Vectorette library is brought together by ligating the Vectorette units to the appropriate DNA fragments which were previously digested. [1] [6] Ligation is the act of binding two things together. [10] A Vectorette unit is only partially not completely double stranded with a mismatched section located in the center of the unit. [11] The reason it is mismatched is to help it avoid Vectorette primers’ attempts at causing it to undergo first strand synthesis. By doing this any priming that is nonspecific is also avoided. [11] This ligation brings together the vectorette which is double stranded and the ends of the restriction fragments which were previously made in the first step. [12] By doing this, the known sequence which is used to prime the PCR reaction at one side is introduced while the other is primed on the genomic sequence which is already known to the user. [12] The third and last step has two parts to it. This is due to there being two primers, the initiating primer (IP) and the Vectorette primer (VP), that act in different stages. During the first part, the IP works on amplifying the primer extension while the VP remains hybridized with the product; thus, any background amplification is not carried out at this stage. However, this changes during the last and following part of PCR as the priming that is performed comes from both the IP and the VP. [6]

Research

A lot of research has been conducted on Vectorette PCR and the applications it has in the field of biology. Scientists used Vectorette PCR to take the transgene flanking DNA and isolate it. They used this technique on the DNA belonging to mice that was next to transgene sections. From this the scientists were able to show that the use of Vectorettes is capable of facilitating the recovery and mapping of sequences in complex genomes. They have also found that Vectorette PCR can help in the analysis of sequences by subvectoretting when PCR products of a large size are the subject at hand. [5]

Other work has looked at developing a method using Vectorette PCR in order to accomplish genomic walking. By using Vectorette PCR, scientists were able to acquire single-stranded DNA which were obtained from PCR products in order to sequence them. From this an approach was identified in which the amplification of sequences which were previously uncharacterized was possible. This research demonstrates how novel sequences can be rapidly developed when only a known sequence of DNA is used to start. [6]

Further research has experimented with the creation of a method that progresses the isolation of microsatellite repeats. By using Vectorette PCR, researchers have found a rapid technique to accomplish this with novel, microsatellite repeats. They have attempted and succeeded in using this technique to isolate an amount of six microsatellite repeats. [13]

Vectorette PCR has also been used to not only identify genomic positions of insertion sequences (IS) but also to map them. Research on this has shed light on a way to complete the typing of microbial stains and the identification and mapping of things like IS insertion sites that reside in microbial genomes. Vectorette PCR proves useful when it comes to rapidly and simply surveying genomes’ IS elements. [14]

Transposable element, transposon, or TE is a variation of genetic elements that is capable of changing its location in a genome by a process called “jumping”. [15] TE display is designed to present the different variations of TE insertion sites which helps to make numerous dominant markers. [16] A problem that arose in the original method was finding a PCR method that was capable of being specific and efficient in its output of the transposon within the genome. [16] Researchers have found a solution for this problem by using Vectorette PCR as the PCR method. Since Vectorette PCR is capable of being specific with its isolation and amplification of genes, this helped with their research and aided in improving the method of TE display by saving both time and costs. [16] The researchers were then able to produce numerous dominant markers with the use of Vectorette PCR that is based on a TE display that is nonradioactive. [16]

Thyroid lymphoma is an illness which leads to the transformation of the lymphocytes belonging to the thyroid into cells of a cancerous nature. [17] Researchers have tested a new method that aids in the diagnosis of this condition. The use of Vectorette PCR was combined with restriction enzyme digestion, and it was found that Vectorette PCR proved to be useful in their study and aided in the diagnosis of thyroid lymphoma. [18]

Researchers have looked into the potential use of Vectorette PCR in the examination of the genes of diseases. They have taken two methods, trinucleotide repeats which are specifically used for the targeting of transcribed regions and Vectorette PCR, to obtain simple sequence repeats or SSRs. [19] It is believed that genetic markers can be made from these SSRs. The outcome from this research is hoped to aid researchers attempt the derivation of genetic markers which are transportable from unknown genomes. Vectorette PCR was used to uncover SSRs which flank the trinucleotide repeat that was targeted for testing. [19] This is also known as TNR or trinucleotide Vectorette PCR. They believe that their TNR method combined with the amplification provided by Vectorette PCR can be used in eukaryotes to create molecular markers that are based on simple repeat sequences. The researchers also think that this method will be of value when attempting to isolate genes that are able to bring about diseases. [19]

Uses

The uses that have been derived from Vectorette PCR are many and have been useful to the science of biology. For example, it gives rise to methods that can help during the outbreaks of diseases by making it easier to subtype pathogens that are similar or closely related. [14] It can also be used to help diagnose certain diseases. [18] Earlier in this page it was noted that Vectorette PCR can give rise to multiple functions that can be performed on novel DNA sequences located near a sequence that is already known. These functions like isolating DNA, amplifying it, and analyzing it are behind the uses for Vectorette PCR. [1] These uses are things like genome walking, DNA sequencing for the termini of Yeast Artificial Chromosomes (YAC) and cosmid inserts, being able to map introns and promoters in genomic DNA and regions with mutations, facilitating the sequencing of clones of a large size, and filling in the gaps that arise during the mapping of genomes. [1]

An intron is a DNA sequence that is flanked by exons and therefore located in between them. [20] It is the region that gets cut out while exons are expressed, and so introns do not affect the code of amino acids. Gene expression can be affected by only a number of intronic sequences. [20] Vectorette PCR has been found to be beneficial when it comes to the characterization of these intronic sequences when they are found to be next to known sequences. [21]

cDNA or complementary DNA is a DNA sequence which is complementary to the RNA that is the template when synthesizing DNA during the reverse transcriptase process. [22] Vectorette PCR that utilizes the primers that originate from cDNA gives rise to a method that is capable of acquiring intron sequences which are located adjacent to exons and aiding in the development of the structure of genes. [23] It is able to achieve this when initializing the process with a sequence of cDNA and a clone of a genome. [23]

Vectorette PCR also gives the user an advantage than if he/she were using other existing technologies. The user will be able to carry out tasks like gene manipulation that is cell-free, Vectorette PCR with minimal material to start with, and performing Vectorette PCR with DNA that needs not be of high purity. These advantages allow the user to save time and resources while increasing the range of DNA that can be targeted. [1]

Chromosome Walking

Chromosome walking can be used for the purpose of cloning a gene. [24] It does this by using the known gene’s markers that are closest and can therefore be used in techniques like isolating DNA sequences and aiding in the sequencing and cloning of the DNA of organisms. Chromosome walking is also useful when it comes to filling in the gaps that may be present in genomes by locating clones that overlap with a library clone end. This means that for chromosome walking to be carried out, it requires a clone library of a genomic format. This is why Vectorette PCR is one of the methods that can be used to create this library for chromosome walking to occur. Vectorette PCR comes in handy when it is necessary to obtain the regions that are both upstream and downstream and flank a sequence that is already known. By obtaining these regions, it provides the library of a genomic format that chromosome walking requires.[ citation needed ]

Yeast Artificial Chromosome

Yeast artificial chromosome or YAC is a DNA molecule that is developed by humans to take the DNA sequences that belong to yeast cells and clone them. [25] Yeast artificial chromosomes can be inserted with fragments of DNA from the organism of interest. Yeast cells will then assimilate the yeast artificial chromosome that contains the DNA from the organism of interest. [25] The yeast cells then multiply in number and this brings about the amplification of the DNA that has been incorporated into it which is then isolated for the purpose of things like sequencing and mapping of the DNA desired i.e. the DNA originally inserted into the yeast artificial chromosome. [25] Vectorette PCR helps with this process by bringing about not only the isolation of the yeast artificial chromosome’s ends but also the amplification of the ends. [26]

Related Research Articles

<span class="mw-page-title-main">Polymerase chain reaction</span> Laboratory technique to multiply a DNA sample for study

The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

A microsatellite is a tract of repetitive DNA in which certain DNA motifs are repeated, typically 5–50 times. Microsatellites occur at thousands of locations within an organism's genome. They have a higher mutation rate than other areas of DNA leading to high genetic diversity. Microsatellites are often referred to as short tandem repeats (STRs) by forensic geneticists and in genetic genealogy, or as simple sequence repeats (SSRs) by plant geneticists.

<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

<span class="mw-page-title-main">Yeast artificial chromosome</span> Genetically engineered chromosome derived from the DNA of yeast

Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of bacterial artificial chromosome

Repeated sequences are short or long patterns of nucleic acids that occur in multiple copies throughout the genome. In many organisms, a significant fraction of the genomic DNA is repetitive, with over two-thirds of the sequence consisting of repetitive elements in humans. Some of these repeated sequences are necessary for maintaining important genome structures such as telomeres or centromeres.

<span class="mw-page-title-main">Library (biology)</span>

In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated.

In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events. It can be formed artificially, using various methods including polymerase chain reactions (PCR) or ligase chain reactions (LCR), or naturally through gene duplication. In this context, amplification refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon. As it refers to the product of an amplification reaction, amplicon is used interchangeably with common laboratory terms, such as "PCR product."

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

Primer walking is a technique used to clone a gene from its known closest markers. 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 or to discover the unknown sequence of a full plasmid or PCR product by designing primers to sequence overlapping sections.

P elements are transposable elements that were discovered in Drosophila as the causative agents of genetic traits called hybrid dysgenesis. The transposon is responsible for the P trait of the P element and it is found only in wild flies. They are also found in many other eukaryotes.

A genomic library is a collection of overlapping DNA fragments that together make up the total genomic DNA of a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. Next, the vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.

In the fields of bioinformatics and computational biology, Genome survey sequences (GSS) are nucleotide sequences similar to expressed sequence tags (ESTs) that the only difference is that most of them are genomic in origin, rather than mRNA.

Synthetic genomics is a nascent field of synthetic biology that uses aspects of genetic modification on pre-existing life forms, or artificial gene synthesis to create new DNA or entire lifeforms.

Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing. This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.

The following outline is provided as an overview of and topical guide to genetics:

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

The versatility of polymerase chain reaction (PCR) has led to modifications of the basic protocol being used in a large number of variant techniques designed for various purposes. This article summarizes many of the most common variations currently or formerly used in molecular biology laboratories; familiarity with the fundamental premise by which PCR works and corresponding terms and concepts is necessary for understanding these variant techniques.

Delitto perfetto is a genetic technique for in vivo site-directed mutagenesis in yeast. This name is the Italian term for "perfect murder", and it refers to the ability of the technique to create desired genetic changes without leaving any foreign DNA in the genome.

Diversity Arrays Technology (DArT) is a high-throughput genetic marker technique that can detect allelic variations to provides comprehensive genome coverage without any DNA sequence information for genotyping and other genetic analysis. The general steps involve reducing the complexity of the genomic DNA with specific restriction enzymes, choosing diverse fragments to serve as representations for the parent genomes, amplify via polymerase chain reaction (PCR), insert fragments into a vector to be placed as probes within a microarray, then fluorescent targets from a reference sequence will be allowed to hybridize with probes and put through an imaging system. The objective is to identify and quantify various forms of DNA polymorphism within genomic DNA of sampled species.

Physical map is a technique used in molecular biology to find the order and physical distance between DNA base pairs by DNA markers. It is one of the gene mapping techniques which can determine the sequence of DNA base pairs with high accuracy. Genetic mapping, another approach of gene mapping, can provide markers needed for the physical mapping. However, as the former deduces the relative gene position by recombination frequencies, it is less accurate than the latter.

References

  1. 1 2 3 4 5 6 7 8 9 10 "The Vectorette System Gene Walking Made Easy Instruction Manual" (PDF). 2019.
  2. ↑ "Polymerase Chain Reaction (PCR)". www.ncbi.nlm.nih.gov. Retrieved 2019-05-11.
  3. ↑ Riley J, Butler R, Ogilvie D, Finniear R, Jenner D, Powell S, Anand R, Smith JC, Markham AF (May 1990). "A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones". Nucleic Acids Research. 18 (10): 2887–90. doi:10.1093/nar/18.10.2887. PMC   330815 . PMID   2161516.
  4. ↑ "vectorette PCR - Terminology of Molecular Biology for vectorette PCR – GenScript". www.genscript.com. Retrieved 2019-05-11.
  5. 1 2 Allen MJ, Collick A, Jeffreys AJ (October 1994). "Use of vectorette and subvectorette PCR to isolate transgene flanking DNA". PCR Methods and Applications. 4 (2): 71–5. doi: 10.1101/gr.4.2.71 . hdl: 2381/4299 . PMID   7580887.
  6. 1 2 3 4 5 Arnold C, Hodgson IJ (August 1991). "Vectorette PCR: a novel approach to genomic walking". PCR Methods and Applications. 1 (1): 39–42. doi: 10.1101/gr.1.1.39 . PMID   1842919.
  7. ↑ "Introduction to the Vectorette System" (PDF). Retrieved 11 May 2019.
  8. ↑ McPherson, M. J. (2000). PCR. Møller, S. G. Oxford: BIOS. p. 235. ISBN   978-0585425306. OCLC   50760423.
  9. ↑ PCR cloning protocols . Chen, Bing-Yuan., Janes, Harry W. (2nd ed.). Totowa, N.J.: Humana Press. 2002. pp.  278. ISBN   0585403341. OCLC   50175114.{{cite book}}: CS1 maint: others (link)
  10. ↑ "Definition of LIGATION". www.merriam-webster.com. Retrieved 2019-05-12.
  11. 1 2 PCR cloning protocols . Chen, Bing-Yuan., Janes, Harry W. (2nd ed.). Totowa, N.J.: Humana Press. 2002. pp.  276. ISBN   0585403341. OCLC   50175114.{{cite book}}: CS1 maint: others (link)
  12. 1 2 Cammack R, Atwood T, Campbell P, Parish H, Smith A, Vella F, Stirling J, eds. (2006-01-01). Oxford Dictionary of Biochemistry and Molecular Biology (2 ed.). Oxford University Press. doi:10.1093/acref/9780198529170.001.0001. ISBN   9780198529170.
  13. ↑ Lench NJ, Norris A, Bailey A, Booth A, Markham AF (June 1996). "Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers". Nucleic Acids Research. 24 (11): 2190–1. doi:10.1093/nar/24.11.2190. PMC   145905 . PMID   8668553.
  14. 1 2 Zhong S, Dean AM (July 2004). "Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR". BMC Microbiology. 4: 26. doi: 10.1186/1471-2180-4-26 . PMC   481064 . PMID   15242519.
  15. ↑ "Transposon | genetics". Encyclopedia Britannica. Retrieved 2019-05-31.
  16. 1 2 3 4 Wang, Jianjun; Miller, Thomas A.; Park, Yoonseong (2006). "Development of multiple dominant markers by using Vectorette PCR-based nonradioactive transposable element display". Molecular Ecology Notes. 6 (3): 642–645. doi:10.1111/j.1471-8286.2006.01403.x. ISSN   1471-8286.
  17. ↑ "Thyroid Lymphoma | Columbia University Department of Surgery". columbiasurgery.org. Retrieved 2019-05-17.
  18. 1 2 Takano T, Asahi S, Matsuzuka F, Hidaka Y, Yoshida H, Miyauchi A (January 2008). "Aspiration biopsy-nucleic acid diagnosis of thyroid malignant lymphoma by vectorette PCR: experience of eight cases". Leukemia Research. 32 (1): 151–4. doi:10.1016/j.leukres.2007.03.019. PMID   17442390.
  19. 1 2 3 Hilario, Elena; Fraser, Lena G.; McNeilage, Mark (2009-03-10). "Trinucleotide Repeats as Bait for Vectorette PCR: A Tool for Developing Genetic Mapping Markers". Molecular Biotechnology. 42 (3): 320–326. doi:10.1007/s12033-009-9157-9. ISSN   1073-6085. PMID   19277911. S2CID   2347805.
  20. 1 2 "Definition of intron - NCI Dictionary of Cancer Terms". National Cancer Institute. 2012-07-20. Retrieved 2019-05-31.
  21. ↑ Rubie, C.; Schulze-Bahr, E.; Wedekind, H.; Borggrefe, M.; Haverkamp, W.; Breithardt, G. (September 1999). "Multistep-Touchdown Vectorette-PCR—A Rapid Technique for the Identification of IVS in Genes". BioTechniques. 27 (3): 414–418. doi: 10.2144/99273bm03 . ISSN   0736-6205. PMID   10489595.
  22. ↑ "Definition of CDNA". www.merriam-webster.com. Retrieved 2019-05-31.
  23. 1 2 Roberts, Roland G.; Coffey, Alison J.; Bobrow, Martin; Bentley, David R. (August 1992). "Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR". Genomics. 13 (4): 942–950. doi:10.1016/0888-7543(92)90005-D. PMID   1505985.
  24. ↑ "Chromosome Walking". Defined Term - A dictionary of defined terms for the legal profession. Retrieved 2019-05-31.
  25. 1 2 3 "Yeast Artificial Chromosome (YAC)". Genome.gov. Retrieved 2019-05-31.
  26. ↑ Kleyn PW, Wang CH, Lien LL, Vitale E, Pan J, Ross BM, Grunn A, Palmer DA, Warburton D, Brzustowicz LM (July 1993). "Construction of a yeast artificial chromosome contig spanning the spinal muscular atrophy disease gene region". Proceedings of the National Academy of Sciences of the United States of America. 90 (14): 6801–5. Bibcode:1993PNAS...90.6801K. doi: 10.1073/pnas.90.14.6801 . PMC   47020 . PMID   8341701.
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