PCR Components

• Critical component of the PCR.

  Primer
• They determine the specificity of the PCR.

  Primer
• The are chemically manufactured on a DNA synthesizer and are 20-30 base pairs long.

  Single-stranded DNA fragments
• Works like primers in vivo and contains sequences complementary to the sites flanking the region on interest.

  Primer
• Hybridization Primer that hybridizes to the target DNA sequence on the OPPOSITE (MINUS) STRAND. Just 5’ to the sequences to be amplified.

  Forward Primer
• Hybridization Primer that hybridizes just 3’ to the sequence to be amplified on the PLUS STRAND.

  Reverse Primer
• Binding to target is affected by?

  Primer Sequence (%GC)

  Length of strand
• Melting temperature of the forward and reverse primer must be similar. So both will hybridize optimally at the same annealing temperature.
• Tm can be adjusted by:

  Increasing the length of the primers Placing the primers in areas with more or fewer Gs and Cs in the template
• It is from nucleotide extraction.

  DNA Template
• Routine clinical analyses require?

  100 ng - 1 ug
• Characteristics of best templates.

  Good Condition

  Free of Contaminants

  No Nicks and Breaks
• Building Blocks of DNA.

  Deoxynucleotide Bases
• What are the deoxyribonucleotide bases?

  dATP (adenine)

  dTTP (thymine)

  dGTP (guanine)

  dCTP (cytosine)
• Usual requirement of Deoxyribonucleotide Bases

  0.1-0.5 mm of each
• First polymerase from Escherichia coli.

  DNA Polymerase
• Thermus Aquaticus, Thermostable, Good fidelity.

  Taq Polymerase
• Thermus thermophilus, also has reverse transcriptase activity, and it is used it RT-PCR (RNA template).

  Tth polymerase
• They provide optimal conditions for enzyme activity, are pH buffers and salts that provide cations.


  PCR buffer
• Provides pH for optimal enzyme activity and accurate amplification and can have accessory components.

  Tris buffer
• PH of Tris buffer?

  8-9.5
• Accessory component of the tris buffer that binds inhibitors and stabilizes the enzyme.

  Bovine Serum Albumin (10 to 100 ug/ml)
• Accessory component of tris buffer that provides reducing conditions that may enhance enzyme activity.

  Dithiothreitol (0.1 mm)
• Accessory component of tris buffer that lower the denaturing temperature of DNA with high secondary structure, thereby increasing the availability of primer binding.

  Formamide (1% to 10%)
• Accessory component/s of tris buffer that may also reduce secondary structure to allow ploymerase extension through difficult areas.

  Triton X-100

  Glycerol

  Dimethyl sulfoxide (1% to 10%)
• They affect denaturing and annealing temperatures?

  Monovalent Cations or

  KCl and (NH4)2SO4
• Longer DNA sequences denature slower (inversely proportional) when?

  There is increased salt concentrations
• Mg2+ is needed by what divalent cations(Mgcl2)?

  DNA polymerase
• 1 NTP=?

  1 Mg atom
• If divalent cations is too low?

  By EDTA/other chelators

  Decreased amplicons
• If divalent cations is too high?

  Increased nonspecific products
• Component of a typical PCR reaction where it directs DNA synthesis to the desired region?

  Oligodeoxynucleotides (0.25 mm each primer)
• Components of a typical PCR reaction that are building blocks that direct to the primers?

  dATP, dCTP, dGTP, dTTP

  (0.2 mm each)
• Component of a typical PCR reaction that is a monovalent cation(salt), for optimal hybridization of primers to template?

  50 mm KCl
• Component of a typical PCR reaction that is a buffer to maintain optimal pH for the enzyme reaction?

  10 mm Tris

  pH 8.4
• Component of a typical PCR reaction that is a divalent cation, required by the enzyme?

  1.5 mm MgCl2
• Component of a typical PCR reaction that is a polymerase enzyme that extends on primers (adds dNTPs)?

  2.5 units polymerase
• Component of a typical PCR reaction that is a sample DNA that is being tested?

  10^2-10^5 copies of template
• First PCRs are?

  Multiple water baths and heat blocks

  Done manually
• It is a machine that rapidly and automatically change to the required temperatures for each step and holds it there for designated periods.

  Thermal Cycler