A primer is a nucleic acid strand, or a related molecule that serves as a starting point for DNA replication. A primer is required because most DNA polymerases, enzymes that catalyze the replication of DNA, cannot begin synthesizing a new DNA strand from scratch, but can only add to an existing strand of nucleotides. This article or section does not cite its references or sources. ... In chemistry, a molecule is an aggregate of two or more atoms in a definite arrangement held together by chemical bonds [1] [2] [3] [4] [5]. Chemical substances are not infinitely divisible into smaller fractions of the same substance: a molecule is generally considered the smallest particle of a pure... This article or section is in need of attention from an expert on the subject. ... 3D structure of the DNA-binding helix-hairpin-helix motifs in human DNA polymerase beta A DNA polymerase is an enzyme that assists in DNA replication. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... The structure of part of a DNA double helix Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions for the development and function of living organisms. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ...

In most natural DNA replication, the ultimate primer for DNA synthesis is a short strand of RNA. This RNA is produced by an RNA polymerase, and is later removed and replaced with DNA by a DNA polymerase. Ribonucleic acid (RNA) is a nucleic acid polymer consisting of nucleotide monomers. ... RNAP from pictured during elongation. ...

Many laboratory techniques of biochemistry and molecular biology that involve DNA polymerases, such as DNA sequencing and polymerase chain reaction (PCR), require primers. The primers used for these techniques are usually short, chemically synthesized DNA molecules with a length about twenty bases. The actual construction of such primers starts with 3'-hydroxyl nucleosides (phosphoramidite) attached to a so-called controlled-pore glass (CPG). The 5'-hydroxyl of the nucleosides is covered dimethoxytrityl (DMT), which prevents the building of a nucleotide chain. To add a nucleotide, DMT is chemically removed, and the nucleotide is added. The 5'-hydroxyl of the new nucleotide is blocked by DMT, preventing the addition of more than one nucleotide to each chain. After that, the cycle is repeated for each nucleotide in the primer. This is a simplified description; the actual process is quite complicated. For that reason, most laboratories do not make primers themselves, but order them by specialized companies. Biochemistry is the study of the chemical processes and transformations in living organisms. ... Molecular biology is the study of biology at a molecular level. ... In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. ... PCR redirects here. ... Nucleoside phosphoramidites are used to synthesise short nucleic acid chains. ...

DNA sequencing is used to determine the nucleotides in a DNA strand. A sequencing method called dideoxy sequencing, also known as chain termination method or Sanger method, uses a primer as a start marker for the chain reaction. The chain termination or Sanger or dideoxy method is a process used to sequence (read the bases) of DNA. It is named after Frederick Sanger who developed the process in 1975. ... The chain termination or Sanger or dideoxy method is a process used to sequence (read the bases of) DNA. It is named after Frederick Sanger who developed the process in 1975. ... The chain termination or Sanger or dideoxy method is a process used to sequence (read the bases) of DNA. It is named after Frederick Sanger who developed the process in 1975. ...

In polymerase chain reaction, primers are used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 50 nucleotides (since DNA is usually double-stranded, its length is measured in base pairs. The length of single-stranded DNA is measured in bases or nucleotides), and they match exactly the beginning and the end of the DNA fragment to be amplified. They anneal (adhere) to the DNA template at these starting and ending points, where the DNA-Polymerase binds and begins the synthesis of the new DNA strand. Annealing, in genetics, means for DNA or RNA to pair by hydrogen bonds to a complementary sequence, forming a double-stranded polynucleotide. ...

Primer design

The choice of the length of the primers and their melting temperature (T_m) depends on a number of considerations. The melting temperature of a primer is defined as the temperature at which 50% of that same DNA molecule species form a stable double helix and the other 50% have been separated to single strand molecules. The melting temperature required increases with the length of the primer. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the temperature required to melt it. Melting temperatures that are too high, i.e., above 80 °C, can also cause problems since the DNA-Polymerase is less active at such temperatures. The optimum length of a primer is generally from 20 to 30 nucleotides with a melting temperature between about 55 °C and 65 °C. There are several ways to calculate the melting temperature of primers. (A, G, C and T are the number of nucleotides in the primer, respectively. [Na⁺] is the concentration of Na⁺ in the PCR vial.) The dissociation of a double-stranded DNA molecule is often referred to as melting because it occurs quickly once a certain temperature has been reached. ... The dissociation of a double-stranded DNA molecule is often referred to as melting because it occurs quickly once a certain temperature has been reached. ... The dissociation of a double-stranded DNA molecule is often referred to as melting because it occurs quickly once a certain temperature has been reached. ...

• "GC"-method : Fast and simple, for primers with more than 13 nucleotides.
  
• "Salt-adjusted"-method : More accurate than GC, for primers with more than 13 nucleotides.
  
• Base-stacking calculation : Most accurate, but complicated

where

ΔH is the enthalpy of base stacking interactions adjusted for helix initiation factors

ΔS is the entropy of base stacking adjusted for helix initiation factors and for the contributions of salts to the entropy

R is the universal gas constant

The free software package primou can calculate the annealing temperature according to the base stacking method. In thermodynamics and molecular chemistry, the enthalpy or heat content (denoted as Δ or ΔH, or rarely as χ) is a quotient or description of thermodynamic potential of a system, which can be used to calculate the useful work obtainable from a closed thermodynamic system under constant conditions. ... Ice melting - classic example of entropy increasing[1] described in 1862 by Rudolf Clausius as an increase in the disgregation of the molecules of the body of ice. ... Molar gas constant (also known as universal gas constant, usually denoted by symbol R) is the constant occurring in the universal gas equation, i. ... PRIMOU is an enhancement of the PRIMO software program developed at the Genome Center at University of Texas Southwestern Medical Center at Dallas to chose primers and to calculate their annealing temperature according to the base stacking method. ...

Also, a primer should not easily anneal with itself or others of its kind, building loops or hairpins in the process. This could hinder the annealing with the template DNA. However, small hairpins are usually unavoidable.

Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. They may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on protein sequence. As several different codons can code for one amino acid, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the amino acid isoleucine 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. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using touchdown PCR. For other meanings of this term, see gene (disambiguation). ... A crab is an example of an organism. ... Peptide sequence or amino acid sequence is the order in which amino acid residues, connected by peptide bonds, lie in the chain. ... RNA codons. ... Phenylalanine is one of the standard amino acids. ... Phenylalanine is one of the standard amino acids. ... Isoleucine is one of the 20 natural amino acids, and is coded for in DNA. Its chemical composition is identical to that of leucine, but the arrangement of its atoms is slightly different, resulting in different properties. ... Adenine is one of the two purine nucleobases used in forming nucleotides of the nucleic acids DNA and RNA. In DNA, adenine binds to thymine via two hydrogen bonds to assist in stabilizing the nucleic acid structures. ... For the similarly-spelled vitamin compound, see Thiamine Thymine, also known as 5-methyluracil, is a pyrimidine nucleobase. ... Adenine is one of the two purine nucleobases used in forming nucleotides of the nucleic acids DNA and RNA. In DNA, adenine binds to thymine via two hydrogen bonds to assist in stabilizing the nucleic acid structures. ... For the similarly-spelled vitamin compound, see Thiamine Thymine, also known as 5-methyluracil, is a pyrimidine nucleobase. ... Cytosine is one of the 5 main nucleobases used in storing and transporting genetic information within a cell in the nucleic acids DNA and RNA. It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached (an amine group at position 4 and a keto group at... RNA codons. ... RNA codons. ... Touchdown PCR or Touchdown style PCR is a method of PCR (polymerase chain reaction) by which degenerate primers will avoid amplifying nonspecific sequence. ...

References

An earlier version of the above article was posted on Nupedia.

External links

• Primer3Plus -- Easy and powerful primer design, based on primer3
• Primer 3 -- Primer3 -- Online primer design software
• FastPCR PCR primer/probe design software
• Primo -- Online primer design
• TM calculation - by bioPHP.org.
• MeSH DNA+primers
• MeSH RNA+primers