In response to many requests, A Primer of Population Genetics, Third Edition, has been dramatically shortened and streamlined for greater accessibility.Designed primarily for undergraduates, it will also serve for graduate students and professionals in biology and other sciences who desire a concise but comprehensive overview of the field with a primary focus on the integration of experimental. The Genetics Society of America (GSA), founded in 1931, is the professional membership organization for scientific researchers and educators in the field of genetics. Our members work to advance knowledge in the basic mechanisms of inheritance, from the molecular to the population level. Online ISSN: 1943-2631.
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The DNA replication fork. RNA primer labeled at top.
A primer is a short single-stranded nucleic acid utilized by all living organisms in the initiation of DNA synthesis. The enzymes responsible for DNA replication, DNA polymerases, are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand.[1] Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis (such as DNA sequencing and polymerase chain reaction) usually use DNA primers, since they are more temperature stable.
- 1RNA Primers in vivo
- 2Uses of synthetic primers
RNA Primers in vivo[edit]
RNA primers are used by living organisms in the initiation of synthesizing a strand of DNA. A class of enzymes called primases add a complementary RNA primer to the reading template de novo on both the leading and lagging strands. Starting from the free 3’-OH of the primer, known as the primer terminus, a DNA polymerase can extend a newly synthesized strand. The leading strand in DNA replication is synthesized in one continuous piece moving with the replication fork, requiring only an initial RNA primer to begin synthesis. In the lagging strand, the template DNA runs in the 5′→3′ direction. Since DNA polymerase cannot add bases in the 3′→5′ direction complementary to the template strand, DNA is synthesized ‘backward’ in short fragments moving away from the replication fork, known as Okazaki fragments. Unlike in the leading strand, this method results in the repeated starting and stopping of DNA synthesis, requiring multiple RNA primers. Along the DNA template, primase intersperses RNA primers that DNA polymerase uses to synthesize DNA from in the 5′→3′ direction.[1]
Another example of primers being used to enable DNA synthesis is reverse transcription. Reverse transcriptase is an enzyme that uses a template strand of RNA to synthesize a complementary strand of DNA. The DNA polymerase component of reverse transcriptase requires an existing 3' end to begin synthesis.[1]
Primer removal[edit]
After the insertion of Okazaki fragments, the RNA primers are removed (the mechanism of removal differs between prokaryotes and eukaryotes) and replaced with new deoxyribonucleotides that fill the gaps where the RNA was present. DNA ligase then joins the fragmented strands together, completing the synthesis of the lagging strand.[1]
In prokaryotes, DNA polymerase I synthesizes the Okazaki fragment until it reaches the previous RNA primer. Then the enzyme simultaneously acts as a 5′→3′ exonuclease, removing primer ribonucleotides in front and adding deoxyribonucleotides behind until the region has been replaced by DNA, leaving a small gap in the DNA backbone between Okazaki fragments which is sealed by DNA ligase.
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In eukaryotic primer removal, DNA polymerase δ extends the Okazaki fragment in 5′→3′ direction, and upon encountering the RNA primer from the previous Okazaki fragment, it displaces the 5′ end of the primer into a single-stranded RNA flap, which is removed by nuclease cleavage. Cleavage of the RNA flaps involves either flap structure-specific endonuclease 1 (FEN1) cleavage of short flaps, or coating of long flaps by the single-stranded DNA binding protein replication protein A (RPA) and sequential cleavage by Dna2 nuclease and FEN1.[2]
Uses of synthetic primers[edit]
Diagrammatic representation of the forward and reverse primers for a standard PCR
Synthetic primers are chemically synthesized oligonucleotides, usually of DNA, which can be customized to anneal to a specific site on the template DNA. In solution, the primer spontaneously hybridizes with the template through Watson-Crick base pairing before being extended by DNA polymerase. The ability to create and customize synthetic primers has proven an invaluable tool necessary to a variety of molecular biological approaches involving the analysis of DNA. Both the Sanger chain termination method and the “Next-Gen” method of DNA sequencing require primers to initiate the reaction.[1]
PCR primer design[edit]
The polymerase chain reaction (PCR) uses a pair of custom primers to direct DNA elongation toward each-other at opposite ends of the sequence being amplified. These primers are typically between 18 and 24 bases in length, and must code for only the specific upstream and downstream sites of the sequence being amplified. A primer that can bind to multiple regions along the DNA will amplify them all, eliminating the purpose of PCR.[1]
A few criteria must be brought into consideration when designing a pair of PCR primers. Pairs of primers should have similar melting temperatures since annealing during PCR occurs for strands both simultaneously, and this shared melting temperature must not be either too much higher or lower than the reaction's annealing temperature. A primer with a Tm (melting temperature) too much higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence. A Tm significantly lower than the annealing temperature may fail to anneal and extend at all.
Additionally, primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of hybridization to a similar sequence nearby. A commonly used method for selecting a primer site is BLAST search, whereby all the possible regions to which a primer may bind can be seen. Both the nucleotide sequence as well as the primer itself can be BLAST searched. The free NCBI tool Primer-BLAST integrates primer design and BLAST search into one application,[3] as do commercial software products such as ePrime and Beacon Designer. Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design by giving melting and annealing temperatures, etc.[4]
Many online tools are freely available for primer design, some of which focus on specific applications of PCR. The popular tools Primer3Plus and PrimerQuest can be used to find primers matching a wide variety of specifications. Highly degenerate primers for targeting a wide variety of DNA templates can be interactively designed using GeneFISHER. Primers with high specificity for a subset of DNA templates in the presence of many similar variants can be designed using DECIPHER. Primer design aims to generate a balance between specificity and efficiency of amplification.[5]
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Selecting a specific region of DNA for primer binding requires some additional considerations. Regions high in mononucleotide and dinucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers should not easily anneal with other primers in the mixture; this phenomenon can lead to the production of 'primer dimer' products contaminating the end solution. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA.
When designing primers, additional nucleotide bases can be added to the back ends of each primer, resulting in a customized cap sequence on each end of the amplified region. One application for this practice is for use in TA cloning, a special subcloning technique similar to PCR, where efficiency can be increased by adding AG tails to the 5′ and the 3′ ends.[6]
Degenerate primers[edit]
Some situations may call for the use of degenerate primers. These are mixtures of primers that are similar, but not identical. These may be convenient when amplifying the same gene from different organisms, as the sequences are probably similar but not identical. This technique is useful because the genetic code itself is degenerate, meaning several different codons can code for the same amino acid. This allows different organisms to have a significantly different genetic sequence that code for a highly similar protein. For this reason, degenerate primers are also used when primer design is based on protein sequence, as the specific sequence of codons are not known. Therefore, primer sequence corresponding to the amino acidisoleucine might be 'ATH', where A stands for adenine, T for thymine, and H for adenine, thymine, or cytosine, according to the genetic code for each codon, using the IUPAC symbols for degenerate bases. Degenerate primers may not perfectly hybridize with a target sequence, which can greatly reduce the specificity of the PCR amplification.
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Degenerate primers are widely used and extremely useful in the field of microbial ecology. They allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in GenBank. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations of the codon sequence.
See also[edit]
- Oligonucleotide synthesis – the methods by which primers are manufactured
References[edit]
- ^ abcdefCox, Michael M. (2015). Molecular Biology: Principles and Practice. 41 Madison Avenue, New York, NY 10010: W. H. Freeman and Company. pp. 221–238, 369–376, 592–593. ISBN9781464126147.
- ^Distinguishing the pathways of primer removal during Eukaryotic Okazaki fragment maturation Contributor Author Rossi, Marie Louise. Date Accessioned: 2009-02-23T17:05:09Z. Date Available: 2009-02-23T17:05:09Z. Date Issued: 2009-02-23T17:05:09Z. Description: Dr. Robert A. Bambara, Faculty Advisor. Thesis (PhD) – School of Medicine and Dentistry, University of Rochester. UR only until January 2010. UR only until January 2010.
- ^Primer-BLAST
- ^'Electronic PCR'. NCBI - National Center for Biotechnology Information. Retrieved 13 March 2012.
- ^Dieffenbach, C W; Lowe, T M; Dveksler, G S (1993). 'General concepts for PCR primer design'. Genome Research. 3: S30-S37. doi:10.1101/gr.3.3.s30. Retrieved 23 June 2017.
- ^Adenosine added on the primer 50 end improved TA cloning efficiency of polymerase chain reaction products, Ri-He Peng, Ai-Sheng Xiong, Jin-ge Liu, Fang Xu, Cai Bin, Hong Zhu, Quan-Hong Yao
External links[edit]
Retrieved from 'https://en.wikipedia.org/w/index.php?title=Primer_(molecular_biology)&oldid=917308031'
Genetics Primer Pdf File
Genetics is the study of genes—the heritable information that contains the codes for proteins and other molecules which form and maintain an organism’s structure and function. In most organisms, these genes are found in strands of deoxyribonucleic acid (DNA) molecules. The specific structure of the DNA (described below) ensures that the genetic information can be passed from one generation to the next, while allowing for some reorganization that results in new variations and, ultimately, evolution.
Although nearly all cells in an organism have the same set of DNA (i.e., genome), the genomes vary among organisms, ensuring that (with few exceptions) each individual is unique. The degree of this variation is a measure of how closely related two organisms are. Thus, the differences among the genomes will be smaller among members of a family than among two completely unrelated persons, and those between related species (e.g., humans and chimpanzees) will be smaller than those between more diverse species (e.g., humans and flies).
Higher organisms are made up of various tissues and organs composed of cells with a range of functions, such as nerve, blood, or muscle cells. Yet all these cells contain the same genome. To achieve the necessary variation in cell structure and function, some DNA portions are “active,” or expressed, in certain cells and at particular developmental stages, leading to the production of different end products. In addition, the environment, to some extent, can influence gene expression, resulting in changes in how the organism functions in, and adapts to, its environment.
What Is DNA?
DNA is a large complex molecule constructed from building blocks called nucleotides, each of which consists of a sugar molecule (deoxyribose) attached to an organic base. There are four organic bases and, accordingly, four different nucleotides called adenosine, cytosine, guanosine, and thymidine, generally referred to by their initials A, C, G, and T. In the cell, the nucleotides are arranged as long strings, with two strings interacting at the organic bases to form a double helix. Moreover, because of the chemical structures of these bases, their interactions are highly specific, so that T nucleotides in one strand only can interact with A nucleotides on the other strand and C nucleotides only can interact with G nucleotides. As a result, the two strands are said to be complementary. This feature is the basis for the ability of the DNA to be duplicated faithfully (at least for the most part) when cells divide so that all cells in an organism carry the same DNA sequence, which also can be passed on to the next generation. However, some variations or errors (i.e., mutations) can occur during this duplication, which lead to the variations that ensure the diversity of individuals within one species and also across species.
How Is Genetic Information Converted Into Proteins?
The genetic information is encoded in the order of the nucleotides. A gene is a particular set of these nucleotides that serves as the blueprint for a specific protein. But how does the cell read this building instruction? When a protein is needed in the cell, the DNA double helix at the corresponding gene briefly splits into single strands. This allows certain proteins that mediate specific chemical reactions (i.e., enzymes) to copy the appropriate DNA strand by bringing in new nucleotides complementary to those in the strand (see figure). This process is called transcription. However, these new nucleotides contain a different sugar (i.e., ribose) and instead of the T nucleotides use a fifth nucleotide called uracil (U). The resulting new strand, which is made up of ribose-containing A, C, G, and U nucleotides, is called a ribonucleic acid (RNA). The RNA is released from the DNA (which then “zips” back up with its complementary strand) and is processed further into a messenger RNA (mRNA) that moves as a single strand out of the nucleus into the cytoplasm. There, other enzymes can bind to the mRNA and bring protein building blocks (i.e., amino acids) together to form chains. This process is known as translation (see figure). The order in which the amino acids are assembled is determined by the sequence of nucleotides in the mRNA, with blocks of three mRNA nucleotides representing one amino acid. The sequential steps of transcription and translation—from DNA through the intermediate mRNA to protein—are the process by which genes are expressed.
Steps 1–3a show the way in which the information on a DNA strand is transcribed into mRNA and then translated into proteins.
SOURCE: Hiller-Sturmhöfel, S.; Bowers, B.J.; and Wehner, J.M. Genetic engineering in animal models. Alcohol Health & Research World 19(3):206–213, 1995.
Variations Among Genes
Variations among genes, known as polymorphisms, lead to the production of different gene products (i.e., proteins) and underlie the unique characteristics of each individual. In general, any given gene is quite similar to the same gene in another organism—in other words, the nucleotide sequence is conserved. For example, the genes that code for the alcohol-metabolizing enzyme alcohol dehydrogenase are of the same size and base sequence in most individuals. However, small differences in the base sequence, affecting as little as one nucleotide, can result in a different protein. In some cases, these modifications still allow the gene to produce a functional protein but with slight variations that may affect its function. Other changes in the nucleotide sequence, however, may result in a nonfunctional or incomplete protein. The ability to conserve the sequences coding for important enzymes clearly is important to cells and organisms.
One important aspect of current genetics research is the identification of groups within populations that carry polymorphisms in various genes, resulting in gene variants called alleles. Identifying these polymorphisms can help scientists to better understand how the functions of these genes and their encoded proteins differ and how they relate to certain diseases or other characteristics. For example, do some people produce a form of alcohol dehydrogenase that is more (or less) efficient than other people? And does this influence their risk of developing alcoholism?
To better understand how genes relate to diseases and other characteristics, the National Institutes of Health created the Human Genome Project, which set out to map the human genome by sequencing the entire DNA sequence found in human cells. (Similar mapping efforts also have been conducted for many other organisms.) This project determined not only that the DNA sequence is highly conserved among humans (about 99.5 percent is the same in all humans) but also that numerous combinations of genetic variants exist that are transmitted together and which are known as haplotypes.
To identify such variations in populations with large numbers of samples, researchers are using genetic probes for specific genes with known sequences. These probes typically are short DNA or RNA sequences complementary to a distinctive portion of the gene of interest. This probe then is marked with, for example, a fluorescent tag so that it can be detected if it interacts with the corresponding complementary DNA sequence in a sample.
This idea of using probes to identify differences in DNA sequences can be expanded into broad-scale studies analyzing numerous gene variants across a number of organisms. These genome-wide association studies (GWAS) allow researchers to identify large numbers of variants and to relate them to different outcomes—for example, those associated with different diseases, such as alcoholism.
Identifying candidate genes of particular significance, either through individual probes or large-scale methods such as GWAS, then allows more detailed study of the particular characteristics and expressions of those genes and their role in disease.
Articles from Alcohol Research : Current Reviews are provided here courtesy of National Institute on Alcohol Abuse and Alcoholism