1. The PCR Buffer

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

3. Monovalent Ions

4. 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

5. Divalent

6. 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

7. Taq and Other Thermostable DNA Polymerases

8. 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

9. 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

11. The Advantages and Disadvantages of Taq over Klenow Fragment DNA Polymerase

12. 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

13. Prevent mispriming of the PCR primer

14. 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

15. Analysis of PCR Amplification Products

16. Gel Electrophoresis

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

19. 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………

22. 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.