BIO203 Midterm Merged Slides Set 2

POLYMERASE CHAIN REACTION

Essentials of Genetics

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. PCRA 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 heat-stable DNA polymerase, such as Taq polymerase

• an enzyme derived from the bacterium Thermus

aquaticus. CONCLUSION: PCR can be performed to amplify DNA. It can be extensively modified to perform a wide array of genetic manipulations.

STEPS OF PCR

THREE STEPS: Denaturation, Annealing, 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 single-stranded 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

EXTENSION: At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's. 72°C is used.

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.

INGREDIENTS OF PCR

MAJOR INGREDIENTS: Microfuge tube, Thermal cycler, DNA template, Primers, Buffer, MgCl2, Distilled H2o, 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.

DNA POLYMERASES: Taq

polymerase, Pfu polymerase, Vent

TEMPLATE CAN BE DNA OR RNA: Template DNA, RNA in case of reverse transcriptase

PRIMERS: Primers are single-stranded 18–30 bp long DNA fragments, Complementary to sequences flanking the region to be amplified.

PRIMERS

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, Nucleotide composition

 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

PRIMERS:

FORMULA FOR CALCULATING MELTING

TEMPERATURE OF PRIMERS: Tm = 4(G+C) + 2(A+T)

DNA POLYMERASES · POLYMERASES: · Extension Rate · (nt/sec) · Source · Taq pol · T. aquaticus · Amplitaq (Stoffel · fragment) · >50 · T. aquaticus · Vent · >80 · Thermococcus · litoralis · Pfu · Pyrococcus furiosus · Tth · (RT activity) · >33 · T. thermophilus

Taq: Thermus aquaticus (most commonly used), Tfl: T.

flavus, Tth: T. thermophilus, Tli: Thermococcus litoralis

& Pfu: Pyrococcus furiosus (fidelity) DNA POLYMERASES: Taq polymerase, Pfu polymerase, Vent polymerase

STANDARD PCR 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, 90–96oC, 20-45 seconds  Annealing temperatureP: Controls specificity of hybridization, 40–68oC, 20-30 seconds  Extension temperature: Optimized for individual polymerases, 70–75oC, 30-45 seconds

TEMPERATURES · Temp · For · Comments

94-60-72 Perfect, long primers Higher temp can be used; maximum annealling temp 94-55-72 Good or perfectly matched primers between 19-24 nt Standard conditions

94-50-72 Adequate primers Allows 1-3 mismatches/20 nt

94-48-68 Poorly matched primers Allows 4-5 mismatches/20 nt

94-45-65 Unknown match, likely poor Primers of questionable quality Amplification takes place as the reaction mix is subject to an amplification program and temperatures. The amplification program consists of a series of 20–50 PCR cycles.

INTERPRETATION OF RESULTS

The PCR product should be of the expected size. Misprimes may occur due to non-specific hybridization of primers.

PRIMERS DIMERS MAY REDUCE

AMPLIFICATION: Primer dimers may occur due to hybridization of primers to each other. BLANK REACTION: Controls for contamination contains all reagents except DNA template. NEGATIVE CONTROL: Controls for specificity of the amplification reaction contains all reagents and a DNA template lacking the target sequence.

Negative Control Positive Control

Patient 1 Patient 2 Patient 3 Molecular Marker Blank Reaction

CONCLUSION: The PCR product should be of the expected size.

TYPES OF PCR

 TYPES: Nested PCR, Multiplex PCR, Touchdown PCR, Sequencespecific PCR, Reversetranscriptase PCR, Long-range PCR, Whole-genome amplification,

RAPD PCR (AP-PCR), Quantitative real-time PCR, Long-range PCR, Whole-genome amplification, RAPD PCR (AP-PCR), Quantitative real-time PCR

NESTED PCR

Two pairs (instead of one pair) of PCR primers are used to amplify a fragment. First pair amplify a fragment similar to a standard PCR. Second pair of primers - nested primers (as they lie / are nested within the first fragment). 2ND PCR PRODUCT IS SHORTER THAN FIRST: Second pair of primers bind inside the first PCR product fragment to allow amplification of a second PCR product. 2ND PCR PRODUCT IS SPECIFIC: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. ADVANTAGE: Very low probability of nonspecific amplification.

MULTIPLEX PCR

Multiplex PCR is a variant of PCR which enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences.

SINGLE REACTION, MANY AMPLICONS: By

targeting multiple sequences at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform.

Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction.

AMPLICON SIZE SHOULD BE DIFFERENT OR

BE LABELED: Amplicon sizes should be different enough to form distinct bands when visualized by gel electrophoresis. LABELED PRIMERS: If amplicon sizes overlap, the different amplicons may be differentiated and visualised using primers that have been dyed with different color fluorescent dyes. PRIMERS PAIRS BE OPTIMIZED: The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR. MULTIPLEX PCR: The use of multiple, unique primer sets within a single PCR reaction to produce amplicons