The PCR Buffer

PCR Buffer pH conditions should be maintained ammonium sulphate help to remove any inhibitory products, such as pyrophosphate, ….. accumulate during PCR amplification.

Monovalent Ions

Monovalent Ions…… stimulate the activity of polymerases Sodium (Na+), potassium (K+) and ammonium (NH+4) ions Potassium ions have an optimum stimulatory effect on PCR DNA polymerases NH+ 4 ions compete for the hydrogen bonds

Divalent

Magnesium Ions…… important ingredient of PCR reaction co-factor for thermostable DNA polymerase activity, stimulating the enzymes optimization is frequently performed in combination with an experiment to determine the optimum dNTP concentration magnesium ion concentration range of between 0.5 and 5 Mm elevated magnesium ion concentrations inhibit PCR……double stranded DNA …actually stabilized …. increasing the risk of nonspecific product amplification

Taq and Other Thermostable DNA Polymerases

DNA Polymerases…… DNA polymerase….the success of PCR Klenow fragment of DNA-dependent DNA polymerase I…. disadvantage 1 lacks a 5¢-3¢ exonuclease activity.... optimum reaction temperature at 37°C… impossible to generate PCR….longer than 400 bp

Taq polymerase “in 1976 Chien described a 94kD thermostable DNA-dependant DNA polymerase derived from a eubacterium called Thermus aquaticus” 3¢-5¢ proofreading domain…. missing

The Advantages and Disadvantages of Taq over Klenow Fragment DNA Polymerase

Advantages… only 1.5 nucleotides per second at 37°C Heat stable … half-life of 130 minutes at a temperature of 92.5°C. Reaction vial not… opened in order to add fresh enzyme … reducing contamination optimum reactivity …. 70°C-80°C…helps to prevent any secondary or tertiary structure High optimum temperature …prevent mispriming of the PCR primer Has increased processivity at elevated incubation temperature. Generates significantly higher yields of amplification products (ten times higher) 60 nucleotides per second at 72°C, but only 1.5 nucleotides per second at 37°C Enzyme is not inhibited by chemical contaminants remaining after nucleic acid extraction, e.g. chloroform Taq enzyme frequently adds an overhanging nucleotide

Prevent mispriming of the PCR primer

Disadvantageous of Taq polymerase Susceptible to proteolytic degradation Taq polymerase inhibited … wide variety of compounds which include:- >2% phenol 1% ethanol, DMSO, EDTA Guanidinium HCl urea agar and agarose. 1% isopropanol >5 mM sodium acetate (NaAc) bromide (CTAB), 0.005% sodium dodecyl sulphate (SDS), >20% formamide, RNase inhibitor blood anticoagulant (polyanetholsulphate) Plant polysaccharides such as glycan, acid mucopolysaccharides, polyphenols, rice starch, pectin, dextransulphate and b-glucans

Analysis of PCR Amplification Products

Gel Electrophoresis

Simple agarose gel electrophoresis polysaccharide agarose (poly d-galactose 3,6-anhydro-l galactose purified from marine algae solid compound at room temperature…. powder

PCR Failures… Smear of amplification products…. may be caused by…. Degraded PCR primers, Contamination from previous PCRs, Excessive quantity of Taq High magnesium ion concentration Number of cycles in the PCR Amount of template DNA DNA added to the PCR mix may be impure or degraded………

Recipe for 2L of 10xTBE 218 g Tris base 110 g Boric acid 9.3 g EDTA Dissolve the ingredients in 1.9 L of distilled water. pH to about 8.3 using NaOH and make up to 2 L.