PCR PRIMER DESIGN SARIKA GARG SCHOOL OF BIOTECHNOLGY DEVI AHILYA UNIVERSITY INDORE INDIA

Transcription

1 PCR PRIMER DESIGN SARIKA GARG SCHOOL OF BIOTECHNOLGY DEVI AHILYA UNIVERSITY INDORE INDIA

2 BIOINFORMATICS Bioinformatics is considered as amalgam of biological sciences especially Biotechnology with computer science and information technology.in bioinformatics, main job is development of biosoftwares. Bioinformatics is an interdisciplinary research area which may be broadly defined as the interface between biological and computational sciences. Bioinformatics involves the solution of complex biological problems using computational tools and systems.

3 Bioinformatics includes the collection, organisation,storage and retrieval of biological information from databases. Before the era of Bioinformatics, only two ways of performing biological experiments were available: within a living organism (so-called invivo) or in an artificial environment (so-called invitro).

4 PCR PRIMER DESIGN Designing primers is the most critical parameter for successful PCR. Selective amplification of nucleic acid molecules, that are initially present in minute quantities, provides a powerful tool for analyzing nucleic acids.the polymerase chain reaction is an enzymatic reaction, which follows relatively simple, predictable and well understood mathematical principles. However the scientist often relies on intuition to optimise the reaction. To make PCR an efficient and cost effective tool, some components of PCR such as Taq DNA polymerase, assay buffer, deoxynucleoside triphosphates (dntps), stabilizing agents, DNA Template and oligonucleotide primers must be considered. Efficacy and sensitivity of PCR largely depend on the efficiency of primers.

5 The ability for an oligonucleotide to serve as a primer for PCR is dependent on several factors including: The kinetics of association and dissociation of primer-template duplexes at the annealing and extension temperatures. Duplex stability of mismatched nucleotides and their location. The efficiency with which the polymerase can recognize and extend a mismatched duplex. DNA template quality or purity is not particularly significant for amplification.

6 SELECTION OF PCR AMPLIFICATION PRIMERS For designing primers following parameters should be taken into consideration: I. Primer Length: a Hard Core Factor Specificity, temperature and time of annealing are at least partly dependent on primer length. The rule-of-thumb is to use a primer with a minimal length that ensures a denaturation temperature of 55-56oC. For general studies, primers of typically nucleotides in length are the best. Primers of nucleotides are accepted as best in being sequence specific if the annealing temperature of the PCR reactions is set within 5oC of the dissociation temperature of primer-template duplex.

7 Longer primers (28-35 nucleotides) are required only to discriminate homologous genes within different species or when a perfect complementary sequence to all the template is not expected. II.Terminal Nucleotides Make a Difference: Both the terminals of the primer are of vital importance for a successful amplification. The 3 -end position in the primer affects mispriming. Runs (3 or more) of C s or G s should be avoided as G+C rich sequence leads to mispriming. The primer should have a stable 5 end and an unstable 3 end. Stretches of A and T are also to be avoided as these will open up stretches of the primer-template complex. A G or C is desirable at the 3 end. This GC clamp reduces spurious secondary bands.

8 III. Melting Temperature (Tm): The optimal melting temperature for primers generally lies in the range of 52-58oC. Both of the oligonucleotide primers should be designed such that they have similar melting temperatures. A good working approximation of this value can be calculated using the formula of Wallace etal (1979), Tm = 2(A+T) + 4(G+C) IV. GC Content: GC% is an important characteristic of DNA and provides information about the strength of annealing. Primers should have a GC contents between 45 and 60 percent. GC contents, melting temperature and annealing temperature are strictly dependent on one another.

9 V. Dimers and False Priming Cause Misleading Results: Annealing between the 3 end of one primer molecule and the 5 end of another primer molecule and subsequent extension results in a sharp background product known as primer dimmer. If the primer binds anywhere else than the target site, the amplification specifically is reduced significantly. This leads to weak output or a smear. When some bases at 3 end of the primer bind to target sequence and achieve favorable chances of extension, it also leads to weak output or a smear. VI. Specificity Primer specificity is at least partly dependent on primer length. It is found that there are many more unique 24 base oligos than 15 base pair oligos. Primers must be chosen so that they have a unique sequence within the template DNA that is to be amplified. A primer designed with a highly repetitive sequence will result in a smear when amplifying genomic DNA.

10 VII. Complementary Primer Sequences: Primers need to be designed with absolutely no intra-primer homology beyond 3 base pairs. If a primer has such a region of self homology, snap back, partially double stranded structures, can occur which will interfere with annealing to the template.

11 SECONDARY STRUCTURE An Important factor to consider when designing a primer is the presence of secondary structures. This greatly reduces the number of primer molecules available for bonding in the reaction. The presence of hairpin loops reduces the efficiency by limiting the ability to bind to the target site. No experimental data is available to support the prediction of the thermodynamic properties of hairpin structures, an important factor to consider when designing a primer. Single stranded nucleic acid sequences may have secondary structures due to the presence of complementary sequences within the primer length e.g. hairpin loops.

12 Retrieving Protein Sequences

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17 More advanced ways to retrieve protein sequences

18 Retrieving a list of related protein sequences

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23 Retrieving nucleotide sequences

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27 BLASTing Protein Sequences

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32 Multiple protein sequence alignment with clustalw

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35 Gene Runner

36 Primer 3 It is a software developed by Rozen ans Skaletsky. It is freely available on Internet. This software is provided by the Whitehead Institute as is and any express or implied warranties, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose are disclaimed. This software is freely available on Internet for designing PCR primers.