UNIT 5. NUCLEIC ACID AMPLIFICATION

Target Amplifications

Involves making copies of a target sequence to such a level that they can be detected in vitro

Target Amplifications

simple procedure where cell processes are done in cell-free systems

Polymerase Chain Reaction

mimics in vivo replication

Kary Mullis

who conceived the idea of amplifying DNA in vitro in 1983 while driving one night on a California highway

Escherichia coli plasmid, pBR322

what spp. was used on the first successful amplification of PCR

because DNA pol. enz. was used to drive the replication, and once it started the replication continued in a chain reaction

why did Mullis called the method “polymerase-catalysed chain reaction”

amplicons

what do we call the DNA sequences produced in PCR

primers

are designed to flank the target sequence from both sides

primer

during the PCR run, the _________ provides the free 3’-OH needed by the RNA polymerase to start elongation

1. denaturation

2. annealing

3. extension/elongation

enumerate the PCR cycle/stages:

90 - 95 °C

denaturation is done at what tempt?

50 - 70 °C

annealing is done at what tempt?

68 - 75 °C

extension/elongation is done at what tempt?

primer sequences

annealing process depend on the ________________________

20-30 base

base length of a primer

1. Primer

2. Template

3. dNTPs

4. DNA polymerase

5. PCR buffer

ENUMERATE: components of PCR cocktail

Primer

attaches to the single-stranded template and provides a free 3’-OH required by DNA pol for nucleic acid elongation

Primer

are specifically designed to be homologous (complementary) to sequences flanking the region to be analyzed.

forward primer

are primers that must bind to the target DNA sequence just 5’ to the sequences intended to be amplified

reverse primer

are primers that must bind just 5’ to the sequence to be amplified on the opposite strand of the DNA

placement of primer

the ____________________ will dictate the size of the amplified product

nature of the primer structure

the ______________________ determines the accuracy if binding to its complementary sequence and not to other sequences

DNA ; RNA

template for PCR can either be _____ or _____

template

a component of the PCR cocktail, which can be single-stranded or double-stranded

• high GC content

• secondary structure

What characteristics of aa template may prove to be more difficult to optimize for amplification

Deoxyribonucleotides

are the building blocks of DNA

dNTPs (deoxyribonucleotide triphosphates)

An equimolar mixture of four dNTPs: adenine, thymine, guanine, and cytosine is used for each run

Taq polymerase

what DNA polymerase is used in PCR?

Thermus aquaticus

Taq polymerase was isolated from the thermophilic bacterium ____________________

PCR buffer

a PCR cocktail component that provides the optimal conditions for enzyme activity

1. potassium chloride (20–100 mM)

2. ammonium sulfate (15–30 mM)

3. other salts of monovalent cations

are important buffer components that affect the denaturing and annealing temperatures of the DNA and the enzyme activity.

salt

an increase in _______ concentration makes longer DNA products denature more slowly that shorter DNA products during the amplification

Magnesium chloride (MgCl₂)

A PCR component that provides Mg²⁺ ions

Magnesium chloride (MgCl₂)

A PCR component that act as a cofactor for DNA polymerase, enabling DNA synthesis and helping primer binding. It also very important for enzyme activity.

False

T or F

Magnesium requirements do not vary for each reaction

True

T or F

The influence of buffer/salt concentrations varies with different primers and template

thermal cyclers

A laboratory instrument used in PCR that automatically changes temperature in cycles (denaturation, annealing, and extension) to amplify DNA.

thermocyclers ; PCR machines

thermal cyclers can also be called ______________ and _____________

thermal cyclers

some have gradient functions so different samples can be heated at different temperatures

Positive control

A PCR sample containing known template DNA that should produce amplification, used to confirm that the PCR reagents and procedure are working properly.

Negative control (PCR)

A PCR sample with no template DNA (usually water instead) that should show no amplification, used to detect contamination or unwanted DNA in the reaction.

PCR-clean up kits

PCR products run on a gel can be excised and used in ___________________

1. DNA is eluted from the gel

2. DNA is precipitated again

what is the process when PCR products are run on a gel?

1. multicomplex PCR

2. RT-PCR

3. qPCR

ENUMERATE: types of PCR modification

Multiplex PCR

uses more than one primer pair in a PCR reaction so that multiple amplifications are primed simultaneously

Multiplex PCR

especially useful in typing or identification analyses

Multiplex PCR

Amplifies multiple DNA targets in one PCR reaction using several primer sets.

1. pathogen typing

2. forensic identification kits

What type of kits contain multiple sets of primers that amplify polymorphic DNA regions

Reverse-transcriptase PCR

A type of PCR that converts RNA into complementary DNA (cDNA) using reverse transcriptase before amplification.

complementary DNA (cDNA)

the resulting double-stranded DNA from reverse-transcriptase PCR is called

reverse transcriptase (RT)

What is the enzyme used in reverse-transcriptase PCR

1. Reverse transcriptase (RT)

2. Oligo dT, random primers, gene-specific primers

3. dNTPs

ENUMERATE: components of reverse-transcriptase PCR

Real-time (Quantitative) PCR

modification of the PCR method where you can directly quantitate amount of PCR products

more ; more ; higher

the _____ amplicons, the ______ fluorophores, the ________ the signal

fluorophores

each round of PCR incorporates _______________ into the amplicons

ethidium bromide (EtBr)

Originally, ___________________was used to visualize DNA in qPCR

SYBR dyes

Fluorescent dyes that bind specifically to double-stranded DNA and emit light when exposed to certain wavelengths, allowing real-time detection and quantification of DNA during qPCR.