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  • Bruno Vellutini 18:30 on 2011/11/29 Permalink
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    Design of oligonucleotide primers 

    Background and advice for designing primers, taken from the same book as the previous post.

    • Specificity is crucial; the longer, the higher specificity.
    • Probability: K = [g/2]^G+C x [(1-g)/2]^A+T — K is expected frequency, g is relative G+C content, GCAT number of each nucleotide.
    • 15 nucleotides would be unique within 3×10⁹ genome, but due to bias in codon usage, repetitive DNA sequences and gene families, they are not.
    • Good idea to scan DNA databases to see if only the wanted sequence is returned.

    Selecting primers

    • Analysis: free of homopolymeric tracts, no secundary structures, not self-complimentary, no significant homology with other sequences.
    • List possible forward/reverse primers with calculating the melting point.
    • Selection of well-matched primers: similar in G+C content, but no more than 3 consecutive nucleotides should be complementary.
    • Refine length and placement of oligonucleotides: 3′-terminal nucleotide is G or C.

    Primer design properties

    Base composition

    • G+C between 40-60%.
    • Even distributions of all four bases along the length of the primer.

    Length

    • Complementary region should be 18-25 nucleotides.
    • Primer pair should not differ by >3bp.

    Repeated and self complementary sequences

    • Inverted or self complementary should not be >3bp in length.
    • Tend to form hairpins and obstruct the annealing.

    Complementary between primer pair

    • 3′ terminal should not bind to any site of the other one.
    • Hybrids formation will compete with the expected amplification.

    Melting temperature

    • Temperature of primers (in pair) should not differ by >5°C.
    • Temperature of amplified product should not differ >10°C.
    • Ensures that denaturation occurs in each cycle.

    3′ termini

    • Crucially should end with G or C.
    • However, GC or CG are not recommended since it increases the probability of forming hairpins.

    Adding sequences to 5′ termini

    • Restriction sites, bacteriophage promoters, etc, commonly added to 5′ end.
    • Does not affect annealing.

    Placement of priming sites

    • Maybe constrained by the location of mutations, restriction sites ,coding sequences, microsatellites, cis-acting elements.
    • When for cDNA best to use sequences that bind to different exons, so that contaminating genomic DNA is easily distinguished.

    Primers for degenerate PCR

    • If short sequence aminoacid only, a pool of degenerate oligonucleotides containing all possible coding combinations can be used to amplify.
     
  • Bruno Vellutini 18:00 on 2011/11/29 Permalink
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    PCR basics 

    This is a summary for the Polymerase Chain Reaction with some typical usage values. Taken from:

    Molecular Cloning: A Laboratory Manual (Third Edition)
    By Joseph Sambrook, Peter MacCallum Cancer Institute, Melbourne, Australia; David Russell, University of Texas Southwestern Medical Center, Dallas
    2001 – 2,344 pp – ISBN 978-087969577-4
    http://www.cshlpress.com/default.tpl?cart=1322569198527814047&fromlink=T&linkaction=full&linksortby=oop_title&–eqSKUdatarq=21

    Chapter 8 — In Vitro Amplification of DNA by Polymerase Chain Reaction

    Components

    Thermostable DNA polymerase

    • Taq polymerase — 0.5-2.5 units per standard 25-50µl reaction (efficiency of ~0.7).
    • Typical contains 2×10¹² to 10×10¹² enzyme molecules.

    Pair of synthetic oligonucleotides (primers)

    • Typical non limiting amount: 0.1-0.5µM (6×10¹² to 3×10¹³ molecules) — enough for 30 cycles of amplification of 1kb segment.

    Deoxynucleoside triphosphates (dNTPs)

    • Equimolar amounts of dATP, dTTP, dCTP, dGTP.
    • Concentration: 200-250 µM of each dNTP (with 1.5mM MgCl2) >> 6-6.5µg of DNA in a 50µl.
    • Higher concentrations >4mM are inhibitory, but could also be lower.
    • Should be stored at -20°C in small aliquots and discarded after second cycle of freezing/thawing.

    Divalent cations

    • dNTPs and oligonucleotides bind to Mg²⁺
    • Molar concentration of cation must exceed molar concentration of phosphate groups (dNTPs + primers).
    • Routinely 1.5 mM Mg²⁺ (>4.5 decreases priming).
    • Optimization by series of 0.5mM-5mM, in 0.5mM increments, and narrowing in 0.2mM.
    • Should not contain EDTA or negative íons.

    Buffer to pH

    • Tris-Cl, pH between 8.3-8.8 at room temp and concentration of 10mM.

    Monovalent cations

    • 50mM KCl for amplifying DNA >500bp.
    • 70-100mM improves for shorter sequences.

    Template DNA

    • Single or double stranded.
    • Closed circular DNA templates are slightly less efficient.
    • If more than 10kb restriction enzymes help (when they do not cleave at the target sequence).
    • Typical 1.0µg of DNA (3×10⁵ gene copies).

    Programming

    Denaturation

    • Partly determined by G+C ratio (higher proportion, higher temperatures).
    • Longer strands take longer times to denaturate.
    • Recommendation: 45 seconds at 94-95°C of DNA 55% or less G+C.

    Annealing of primers

    • Critical temperature!
    • If too high, poor annealing. If too low, nonspecific annealing will occur.
    • Usually 3-5°C lower than the calculated melting temperature for primer/template dissociation.
    • Trial with 2°C to 10°C degrees below melting temperature OR touchdown PCR.

    Extension of primers

    • Near optimal temperature of the polymerase enzyme, Taq is 72-78°C with ~2000 nucleotides/minute.
    • 1 minute for 1000bp.

    Number of cycles

    • Depends on the number of copies of template DNA.
    • 30 cycles with 10⁵ copies of target.

    Inhibitors

    • Contaminants of the template DNA are normally the culprits for problems in amplification.
    • Examples: proteinase-k, phenol, EDTA, ionic detergents, heparin, polyanions, hemoglobin, bromophenol blue, xylene cyanol.
    • Solution: cleanup by dialysis, ethanol precipitation, extraction with chloroform and/or chromatography.

    Contamination

    • Common problem with exogenous DNA.
    • Amplification product appearing in the negative controls (without template DNA).
    • Easier to discard all solutions, reagents and recipients and decontaminate instruments, than trying to find the source.
    • Keep traffic in the PCR lab area to a minimum.
    • Wear gloves and change them frequently; use face masks and head caps.
    • Different set of reagents for PCR.
    • Centrifuge microfuge tubes with reagents before opening in the flow hood.
    • Dilutions of DNA at the bench and take needed amounts to PCR.
    • Do not take tubes with amplified DNA to PCR area.

    For basic PCR protocol (and troubleshooting tips) see page 8.18.

    Reverse transcriptase-PCR

    • Amplify cDNA sequences from mRNA.
    • First enzimatic conversion of RNA to single-stranded cDNA by oligodeoxynucleotide (oligo(dT)) primer binding to poli-A mRNA tail and extended by reverse transcriptase DNA polymerase (which will be amplified by the PCR).
    • When possible primers should bind to different exons of the RNA.
    • Positive and negative controls should be done (eg, negative: without template, positive: standard synthetic RNA from a mutated DNA (trickier).

    Rapid amplification of 5′ cDNA ends

    • Sequence specific primer binds to RNA and leads to the first strand of cDNA.
    • RNA and first primers are removed and a homopolymeric tail is added to the 3′ end of the cDNA (with terminal transferase).
    • Use oligo(dT) to prime the second strand of cDNA (at poly-A tail).
    • Use a sequence specific and a oligo(dT) primer to amplify the double-strand cDNA.
    • Finish by cleaving the adaptors with restriction enzymes.

    Rapid amplification of 3′ cDNA ends

    • Oligo(dT) adaptor primer is annealed to mRNAs and first-strand synthesis is achieved with reverse transcriptase and dNTPs.
    • Sequence specific primer executes the second-strand synthesis.
    • Amplification continues with sequence specific primers and complementary primer to the adaptor sequence.

    Rapid characterization of cloned DNA in prokaryotic vectors

    • Transformed bacterial cells are picked from colonies and transferred to PCR mixtures with primers, but without Taq.
    • Reaction mixtures are boiled to liberate template DNA and inactivate nucleases and proteases.
    • Taq is then added and 30 cycles of standard PCR is run.
    • Successful amplification yields a DNA fragment whose size can be estimated with electrophoresis and identity confirmed by sequencing.
     
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