BIO203 Handouts 2022-23

BIO-203 MID TERM · LECTURE 1 TO 39 · Handouts · 1-POLYMERASE CHAIN REACTION · PCR

•  A technique widely used in Molecular Biology and Biotechnology.
•  Its name is from one of its key component DNA polymerase.
•  As PCR progresses, DNA generated is itself used as template for replication.

PCR- A CHAIN REACTION

•  This sets in motion of a chain reaction in which the DNA template is exponentially amplified.

HEAT STABLE DNA POLYMERASE

•  During PCR, a heatstable DNA polymerase, such as Taq polymerase – an enzyme derived from the bacterium Thermus aquaticus.
•  PCR can be performed to amplify DNA.
•  It can be extensively modified to perform a wide array of genetic manipulations.

2-STEPS OF PCR

THREE STEPS

I. Denaturation II. Annealing III. Extension

INITIALIZATION STEP

•  This step consists of heating the reaction to a temperature of 93°C - 96°C, which is held for 1-10 minutes.

DENATURATION

•  First regular cycling event and consists of heating the reaction to 93°C - 98°C for 20- 45 seconds.
•  It causes melting of DNA template yielding single strands of DNA.

ANNEALING

•  The reaction temperature is lowered to 40-65°C for 20-40 seconds.
•  Annealing of the primers to the singlestranded DNA template.

EXTENSION

•  The temperature at this step depends on the type of DNA polymerase used.
•  Taq polymerase has its optimum activity temperature at 70- 80°C.
•  Commonly 72°C is used.
•  At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's.

FINAL ELONGATION

•  Single step is performed at a temperature of 70- 74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

FINAL HOLD TEMPERATURE

•  This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.

3-INGREDIENTS OF PCR

MAJOR INGREDIENTS

•  Microfuge tube
•  Thermal cycler
•  DNA template
•  Primers
•  Buffer
•  MgCl2
•  Distilled water
•  Deoxynucleotide triphosphates
•  DNA polymerase

MICROFUGE TUBES

These are small cylindrical plastic tubes with conical bottoms.

THERMOCYCLER

The thermocycler works on the principle of Peltier effect, which raises and lowers the temperature of the block in a pre-programmed manner.

TEMPLATE CAN BE DNA OR RNA

•  Template DNA
•  RNA in case ofreverse transcriptase

PRIMERS

•  Primers are singlestranded 18–30 bp long DNA fragments
•  Complementary to sequences flanking the region to be amplified.

PRIMERS CAN BE SPECIFIC OR RANDOM

•  Primers determine the specificity of the PCR reaction.
•  Distance between the primers binding sites will determine the size of PCR product.

FEATURES OF PRIMERS

•  Types of primersrandom or specific
•  Primer length
•  Annealing temperature
•  Specificity
•  Nucleotidecomposition

PRIMERS

•  Avoid inter-strand homologies
•  Avoid intra-strand homologies
•  Tm of forward primer = Tm of reverse primer
•  G/C content of 20–80%; avoid longer than GGGG
•  Product size (100–700 bp)
•  Target specificity

FORMULA FOR CALCULATING MELTING TEMPERATURE OF PRIMERS

•  Tm = 4(G+C) + 2(A+T)
•  Taq: Thermus aquaticus (most commonly used)
•  Tfl: T. flavus
•  Tth: T. thermophilus
•  Tli: Thermococcus litoralis
•  Pfu: Pyrococcus furiosus (fidelity)

6-STANDARD PCR REACTION

STANDARD REACTION

•  0.25 mM each primer
•  0.2 mM each dATP, dCTP, dGTP, dTTP
•  50 mM KCl
•  10 mM Tris
•  1.5 mM MgCl2
•  2.5 units polymerase
•  102 - 105 copies of template
•  50 ul reaction volume

PCR TEMPERATURES

Denaturation temperature

•  Reduces double stranded molecules to single stranded

Annealing temperature

•  Controls specificity of hybridization