User:MicailaSpisak/sandbox

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<figure class="mw-halign-right" role="presentation">[./File:DNA_replication.svg ]<figcaption>The DNA replication fork. RNA primer labeled at top.</figcaption></figure> A primer is a strand of short nucleic acid sequences (generally about 10 base pairs) that serves as a starting point for

nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand
.

In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of RNA (which can be made de novo).

Many of the laboratory techniques of

oligonucleotides, with a length of about twenty bases. They are hybridized
to a target DNA, which is then copied by the polymerase.

Mechanism in vivo

Further information: DNA replication § The replication fork

The lagging strand of DNA is that strand of the DNA double helix that is orientated in a 5' to 3' manner. Therefore, its complement must be synthesized in a 3'→5' manner. Because

OH
groups on the RNA primers to synthesize DNA in the 5'→3' direction.

The RNA fragments are then removed by

deoxyribonucleotides are added to fill the gaps where the RNA was present. DNA ligase
then joins the deoxyribonucleotides together, completing the synthesis of the lagging strand.

Primer removal

In eukaryotic primer removal, DNA polymerase δ extends the Okazaki fragment in 5' to 3' direction, and when it encounters 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 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.[1]

This mechanism is a potential explanation of how the

reverse transcription of its RNA. However, the HIV-encoded reverse transcriptase has its own ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that copies the sense cDNA strand into antisense DNA to form a double-stranded DNA intermediate.[2]

Uses of synthetic primers

Diagrammatic representation of the forward and reverse primers for a standard PCR
For the organic chemistry involved, see Oligonucleotide synthesis. For possible methods involving primers, see Nucleic acid methods.

DNA sequencing is used to determine the nucleotides in a DNA strand. The Sanger chain termination method of sequencing uses a primer to start the chain reaction.

In PCR, primers are used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 30 (usually 18–24[3]) nucleotides, and they need to match the beginning and the end of the DNA fragment to be amplified. They direct replication towards each other – the extension of one primer by polymerase then becomes the template for the other, leading to an exponential increase in the target segment.

It is worth noting that primers are not always for DNA synthesis, but can in fact be used by viral polymerases, e.g. influenza, for RNA synthesis.

PCR primer design

Pairs of primers should have similar melting temperatures since annealing in a PCR occurs for both simultaneously. A primer with a Tm (melting temperature) significantly higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence, while Tm significantly lower than the annealing temperature may fail to anneal and extend at all. It is ideal to have at least 50% C-G content in each primer and it is ideal for the C-G content of both the forward and reverse primer to be similar to ensure that they have similar annealing temperatures. A higher C-G content helps the primer anneal strongly to the template DNA. The 3' end of the primer should end in a C-G, this is called the G/C clamp and helps ensure the primer binds well at the end of the primer where the DNA polymerase begins synthesis. There are three different primers commonly used for priming cDNA synthesis from RNA: Specific Priming, Oligo-dT priming, and Random Priming

Primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of mishybridization to a similar sequence nearby. A commonly used method is

Beacon Designer. Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.[5]

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.

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 (either other copies of same or the reverse direction primer); this phenomenon can lead to the production of 'primer dimer' products contaminating the mixture. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA.

When designing a primer for use in TA cloning, efficiency can be increased by adding AG tails to the 5' and the 3' end.[6]

The reverse primer has to be the reverse complement of the given cDNA sequence. The reverse complement can be easily determined, e.g. with online calculators.[7]

Degenerate primers

Main article:
Degenerate bases

Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical primers. They may be convenient if the same

touchdown PCR
.

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.

There are a number of programs available to perform these primer predictions;

  • CODEHOP can be run on a server though the server is no longer supported. It uses the block format.
  • HYDEN is an executable that runs on windows through command prompt.
  • iCODEHOP also uses the block format but has been taken down.
  • FAS-DPD (the most recent) has to be run through a Java operating system (JNODE) that can be set up in a virtual machine(VM).

See also

References

  1. ^ 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.
  2. ^ Doc Kaiser's Microbiology Home Page > IV. VIRUSES > F. ANIMAL VIRUS LIFE CYCLES > 3. The Life Cycle of HIV Community College of Baltimore County. Updated: Jan., 2008
  3. ISBN 978-1-84593-478-1
    . Specificity is influenced by the length of the primers and typically primers between 18–24 nucleotides are suitable for PCR.
  4. ^ Primer-BLAST
  5. ^ "Electronic PCR". NCBI - National Center for Biotechnology Information. Retrieved 13 March 2012.
  6. ^ 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
  7. ^ Reverse Complement Calculator

External links

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