PCR-Touchdown

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Dr. MAHMOUD BEREKAA Molecular Microbiology & Biotechnology PCR & Touchdown-PCRBasics & Applications

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PCR = POLYMERASE CHAIN REACTION PCR amplification is achieved by using oligonucleotide primers

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These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence PCR will allow a short stretch of DNA (usually fewer than 3000 bp) to be amplified to about a million fold so that one can determine its size, nucleotide sequence, etc The particular stretch of DNA to be amplified, called the target sequence

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The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template This results in the synthesis of new DNA strands which are complementary to the parent template strands These new strands have defined 5' ends (the 5' ends of the oligonucleotide primers), whereas the 3' ends are potentially ambiguous in length.

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Primer Design How should I select a set of primers to use for PCR? Some General points: Try to keep the primer 50% G-C Try to avoid Gs and Cs at 3' end of the primers. This may increase the chance of forming primer dimers. Avoid self-annealing regions within each primer. Compute Tm as sum of 4 oC for G/C and 2 for A/T, then subtract 5 oC from this value and that is our annealing temperature.

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Naturally, the annealing temp will be that of the primer with the lower value. Differences of 4-6 oC do not seem to affect yield of PCR. Ideally you would like the Tm for each primer to match and be within the 70-75 OC range. A good practice is to check the target DNA sequence if it is known for mispriming areas. A quick check scanning the sequence of vector for approximately 70% and above homology regions can help prevent obtaining multiple contaminating bands in your PCR. Use of a computer program may help eliminate the use of a poorly designed pair of primers.

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The oligonucleotide directed synthesis of daughter DNA strands can be repeated if the new duplex is denatured (by heating) and additional primers are allowed to anneal (by cooling to an appropriate temperature). The steps of: 1. Template denaturation 2. Primer annealing 3. Primer extension comprise a single "cycle" in the PCR amplification methodology

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After each cycle newly synthesized DNA strands serve as templates in the next cycle

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Materials: sterile water 10X amplification buffer with 15mM MgCl2 10 mM dNTP 50 uM oligonucleotide primer 1 50 uM oligonucleotide primer 2 5 unit/ul Taq Polymerase template DNA (1 ug genomic DNA, 0.1-1 ng plasmid DNA) in 10 ul mineral oil 1. Combine the following for each reaction (on ice) in a 0.5 ml tube: 10X PCR buffer 10 ul Primer 1 1 ul Primer 2 1 ul dNTP 2 ul template DNA and water 85.5 ul Taq Polymerase 0.5 ul

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Prepare a control reaction with no template DNA and an additional 10 ul of sterile water. Add 70-100 ul mineral oil (or 2 drops of silicone oil) to each reaction. Place tubes in a thermal cycler preheated to 94oC. Run the following program: 94 OC 1 min 55 OC 1 min or annealing temperature appropriate for particular primer pair 72 OC 1 min (if product is <500 bp), 3 min (if product is >500 bp) for 30 cycles. Program a final extension at 72oC for 7 min. Electrophorese 10 ul on an agarose gel.

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What kinds of programs are available for designing PCR primers? PrimerGen searches strings of amino acid residues in order to reverse-translate oligonuclotide primers of a desired range of lengths and maximum number of degeneracy. Primer (Stanford) Sun Sparcstations only Amplify, Bill Engels (Macintosh only), is for use in designing, analyzing, and simulating experiments involving the polymerase chain reaction (PCR). You can obtain your copy of Amplify here PrimerDesign 1.04, a DOS-program to choose primer for PCR or oligonucleotide probes. See also the

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PrimerDesign Welcome Page. PC-Rare, a new software by R. Griffais, which uses a rare octamer at the 3' termini of the primer. This powerful (but user friendly) software is available for Macintosh and Windows environment. Primer Design, a Java applet by Luca Ida Giovanni TOLDO. CODEHOP, PCR primers designed from protein multiple sequence alignments (Local mirror site at WIS). Primer3, an online service to pick PCR primers from nucleotide sequence. (Local mirror site at WIS). NetPrimer (PREMIER Biosoft International). NetPrimer combines the latest primer design algorithms with a web-based interface allowing the user to analyze primers over the internet.

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Under suboptimal conditions Multiple undefined and unwanted products are produced No product amplified Changes in the following factors: Mg2+ concentration Buffer concentration Cycling conditions (annealing temperature) Interdependent variables: dNTP, chelater Mg2+ increase in its concentration decreases the concentration of free Mg2+ that influence polymers function For optimization

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Touchdown PCR Allowed the use of a single reaction tube Instead of multiple reaction tubes with different reagent concentrations and / or cycling parameters. PCR, Allow to run under cycling conditions favor amplification of desired amplicon & prevent primer-dimer and artificial amplicons. Touchdown PCR

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The annealing-segments temperature allowed to run initially above the suspected Tm and gradually declines to falls below Tm The high temperature above Tm ensure that the first primer-template hybridization events allow product of greatest complelementarity (target amplicon). Deacreasing Tm to nonspecific hybridization will not affect the target amplicon and it can compete any other nonspecific products.

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When degree of identity between primer and template is UNKNOWN [ when primers are designed on bases of amino acid sequence] In case of Amplification of DNA from one species using primers with identity to a homologous segment of another species. In These cases mismatches between primer and template may lowered Tm of target amplicon enough to approach those of the spurious priming sites.

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Degenerate primers with multiple base variation or inosine residues are often used in such cases But greater variety of sequences in the first case and the relaxed stringency in the latter case might tend to increase the chances of nonspecific priming.

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