PCR TECHNIQUES

Thermocycler

Machine that changes temperature, used in polymerase-catalyzed chain reaction

Steps in PCR

1. Denaturation

2. Annealing

3. Extension

Amplification Technique

Method that generates enough copies of single gene sequence (increases yield)

Kary Mullis

• California (1983)

• First to use the PCR

Sickle Cell Anemia

First application for diagnosis

Escherichia coli plasmid pBR322

First successful amplification

beta-hemoglobin / beta-globin chain

In sickle cell anemia, what is examined is?

DNA polymerase

Drives the replication and to catalyze the addition of nucleotides to the growing strand with a use of primer that adds subsequent bases

PCR Components

1. Primers

2. DNA Template

3. Deoxyribonucleotide Bases

4. DNA Polymerase

5. PCR Buffer

6. PCR Controls

7. Thermal Cyclers

Primers

Most critical component as it determines the specificity of the entire sequence to amplify

• ssDNA with 20—30 base pairs in length

Melting temperature (Tm)

Determines the starting point of the primer

Forward Primer

Primer set that binds to the leading strand

Reverse Primer

Primer set that binds to the lagging strand

Non-specific binding may occur

What will happen if the melting temp is too low?

Flanking the Region

Mode of binding that does not bind directly in the target but at the sides

DNA Template

dsDNA or ssDNA derived from patient’s sample or microorganism causing infection

• 100ng—1ug of DNA

False

(T/F): Templates with high G-C content and tertiary structures may encounter difficulty in amplification and may require modifications

Deoxyribonucleotide Bases

These are what’s being added to the growing strand of the DNA chain

• Standard procedures require 0.1—0.5 mM concentrations

Nucleotide Triphosphate

Building blocks of DNA

• dATP - deoxyadenosinetriphosphate

• dGTP - deoxyguanosinetriphosphat

• dTTP - deoxythymidinetriphosphate

• dCTP - deoxycytidinetriphosphate

Thermus aquaticus

The thermostable enzyme “Taq polymerase” is from?

Thermus thermophilus

The thermostable enzyme “Tth polymerase” is from?

Stoffel Fragment

Modified Taq polymerase that lacks N-terminal 289 amino acid

• Ideal for amplifying regions with high G-C content

PCR Buffer

Provides optimal condition for enzyme activity

False

(T/F): Increasing salt concentration makes shorter DNA products denature more slowly than longer DNA products

True

(T/F): Increasing salt concentration makes longer DNA products denature more slowly than shorter DNA products

Potassium Chloride

PCR buffer that uses 20—100 mM concentration

Ammonium Sulfate

PCR buffer that uses 15—30 mM concentration

Magnesium Chloride

PCR buffer that uses 1—4 mM concentration

Tris-HCl

PCR buffer that uses 10mM concentration with a pH of 8—9.5 for proper buffering

Tris-HCl.

Most important and commonly used buffer in PCR.

Bovine Serum Albumine

Accessory buffer that uses 10-100 ug/mL concentration

Dithiothreitol (DTT)

Accessory buffer that uses 0.01 mM concentration

Formamide

Accessory buffer that uses 1—10% concentration

Chaotropic Agents

• Triton X-100

• Glycerol

• Dimethyl sulfoxide

Dimethyl sulfoxide

Chaotropic agent that uses 1—10% concentration

Magnesium ions

By giving _____, PCR buffers can provide optimal condition for enzyme activity

PCR Controls

Essential for maintaining and ensuring accuracy of the assay

Positive Control

Ensures that the:

1. Enzyme is active

2. Buffer is optimal, and

3. Primers are priming right sequences

Negative Control w/o DNA

Ensures that the reaction mix is not contaminated

Negative Control w/ DNA that lacks target sequence

Ensures that the primers are not annealing to unintended sequences of DNA

Contamination Control / Reagent Blank

Other term for negative control w/o DNA

Negative Template Control

Other term for negative control w/ DNA

Distilled Water

Content of negative control w/o DNA

Thermal Cyclers

Designed to ramp through the required incubation temperature rapidly and automatically in several cycles

Amplicons

These are the PCR products or the copies of DNA

True

(T/F): Within 1—2 hours, PCR can produce millions of amplicons

Amplify

The real advantage of the PCR is the ability to _____ specific targets

Denaturation

In this PCR step, dsDNA is split into 2 ssDNA

• Temp: 90—96° C

• Time: 20—60 seconds

94—96° C

Optimal temp for denaturation

Annealing

Most critical step for the specificity of the PCR; where attachment of primers happens to the strand to be amplified

• Temp: 50—70° C

• Time: 20—90 seconds

Extension

In this PCR step, DNA polymerase catalyzes the formation of phosphodiester bond to replicate DNA

• Temp: 68—75° C

• Time: 10—60 seconds

68—72° C

Optimal temp for extension