PCR Techniques Overview

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Questions and Answers

In ______ PCR, multiple targets are amplified in a single reaction.

Multiplex

Reverse transcriptase PCR converts ______ into complementary DNA.

mRNA

Nested PCR is designed to decrease ______ binding.

non-specific

In ______ PCR, known sequences are used to identify unknown sequences.

Inverse Signup and view all the answers

Overlap PCR is used for joining more PCR products through ______.

splicing Signup and view all the answers

Amplification Fragment Length Polymorphism (AFLP) is a technique for selective amplification of ______ fragments.

restriction Signup and view all the answers

PCR can be utilized for prenatal diagnosis by amplifying DNA from single ______ cells.

embryonic Signup and view all the answers

One application of PCR is the identification of a person based on few skin ______ left behind.

cells Signup and view all the answers

Primers must not bind each other to avoid ______ complementarity.

interstrand Signup and view all the answers

The ideal GC content for primers is between ______% and 60%.

40 Signup and view all the answers

The melting temperature (Tm) of primers should be between ______°C and 70°C.

45 Signup and view all the answers

If the annealing temperature (Ta) is too high, it may cause insufficient primer-template ______.

hybridization Signup and view all the answers

Primers should ideally be ______ to 30 base pairs in length.

18 Signup and view all the answers

Primers should end on a ______ or C or GC of CG to enhance priming efficiency.

G Signup and view all the answers

The optimal annealing temperature for a primer pair can be calculated using the formula: ______ = 0.3 x (Tm of primer) + 0.7 x (Tm of product) - 25.

TaOpt Signup and view all the answers

A type of primer that binds a variety of DNA templates is known as a ______ primer.

universal Signup and view all the answers

PCR products are analyzed using ______ electrophoresis.

gel Signup and view all the answers

The agarose powder is dissolved with boiling ______.

buffer Signup and view all the answers

DNA molecules migrate towards the positive electrode due to their ______ charge.

negative Signup and view all the answers

The size of DNA fragments is measured in ______.

base pairs Signup and view all the answers

Oligonucleotide ______ are essential when running PCR.

primers Signup and view all the answers

Primers must be complementary to the template ______.

strand Signup and view all the answers

DNA fragments move faster through the gel if they are ______.

small Signup and view all the answers

The migration of DNA molecules is inversely proportional to the logarithm of their ______.

lengths Signup and view all the answers

The length of oligonucleotide primers usually ranges from ______ nucleotides.

18-30 Signup and view all the answers

If primers are too ______, they may lead to non-specific amplification.

short Signup and view all the answers

Study Notes

PCR Variations

PCR refers to polymerase chain reaction, a technique used to amplify specific DNA segments.
There are various PCR variations for different applications.

Multiplex PCR

Amplifies multiple targets in a single reaction.
Enables simultaneous analysis of multiple DNA sequences.

Nested PCR

Two consecutive PCR reactions for increased specificity.
The first reaction produces a product that acts as a template for the second, minimizing non-specific binding.
Applications include amplifying targeted sequences in small genomes and degraded DNA.

Inverse PCR

Utilizes known sequences to identify unknown sequences.
Used to determine retroviruses and transposons that integrate randomly into genomic DNA.
Employs restriction enzymes for DNA cutting.

Reverse Transcriptase PCR (RT-PCR)

Detects cell-specific gene expression by converting mRNA to cDNA.
mRNA is the starting material, converted to cDNA using reverse transcriptase.
cDNA is then amplified using conventional PCR.

Overlap PCR/Overlap Extension PCR

Joins multiple PCR products together.
Involves splicing of DNA molecules.
Applications: cloning complex fragments, editing cloned genes, and fusing gene elements.

Amplification Fragment Length Polymorphism (AFLP)

Selectively amplifies restriction fragments from digested genomic DNA.
Polymorphism in different primers aids in detecting variations between DNA samples.
Highly sensitive, used for microsatellite analyses.

Some Uses of PCR

Identification of individuals using minimal DNA samples.
Evolutionary research by amplifying DNA from ancient specimens.
Amplification of DNA from single embryonic cells for prenatal diagnosis.
Disease diagnosis, like HIV and COVID-19.
Basic research on gene structure and genomes.

Analysis of PCR Products: Gel Electrophoresis

Analytical procedure separating DNA fragments by size.
Components:
- PCR product (amplified DNA)
- DNA ladder (molecular marker)
- Agarose powder
- Gel stain
- Loading dye
- Gel and electrophoresis buffers (TBE or TAE)
- Electrophoresis set.

Agarose Gel Electrophoresis

Separates DNA fragments based on size using electric current.
Determines DNA molecule size, ranging from 100 to 30,000 base pairs (bps).

Making the Agarose Gel

Agarose powder dissolves in boiling buffer (TBE or TAE).
Liquid mixture is poured into a casting tray to solidify.
Wells are formed by inserting a comb into the liquid before it solidifies.

Loading DNA and Running the Gel

DNA (PCR product) is mixed with loading dye and loaded into wells.
Gel is placed between positive and negative electrodes.
Voltage and time are selected.
DNA (negatively charged) moves towards the positive electrode (anode).

DNA Molecular Sizes

Smaller fragments move faster through the gel.
Larger fragments move slower.
Migration of DNA is inversely proportional to the logarithm of length (bps).

Primer Design

Oligonucleotide primers are essential for PCR.
Primers must be complementary to the target region/strand.
Primers must amplify unique sequences, unless fingerprinting is desired.
Primers are chemically synthesized.

Primer Design: Length

Typically 18-30 nucleotides long.
Affects primer specificity and annealing efficiency.
Too short: low specificity, leading to non-specific amplification.
Too long: reduces template-binding efficiency, increasing hairpin formation.

Primer Design: Composition

Must not bind each other (interstrand complementarity).
GC content between 40-60% (ideal 50-60%).

Primer Design: Melting Temperature (Tm)

The temperature at which 50% of DNA duplex becomes single-stranded.
Factors:
- Primer length
- Base composition
- Primer concentration
Simple formula for Tm:
- For shorter than 18 bases: Tm = 2(A+T) + 4(G+C)
- For longer primers: Tm = 64.9 + 41(G+C-16.4)/(A+T+G+C)

Primer Design: Annealing Temperature (Ta)

Temperature at which primers bind to complementary DNA regions.
Depends on primer length and base composition.
Ta = 5°C below the lowest Tm of the primer pair.
Optimal Ta:
- Too high: low PCR yield due to insufficient hybridization.
- Too low: non-specific products.
- TaOpt = 0.3 x (Tm of primer) + 0.7 x (Tm of product) - 25

Primer Design: Properties

Length: 18-30 bp
GC content: 40-60%
Tms: between 45-70°C, within 5°C of each other
3’ end: G or C or GC or CG to prevent "breathing" and increase efficiency.
Avoid complementary regions and runs of 3 or more Cs or Gs at the 3’ end.

Primer Design Tools

Online tools assist in primer creation based on a specific DNA sequence.
Examples: Primer Blast, Eurofins Genomics PCR Primer Design Tool, Primer3web.

Different Types of Primers used in PCR

Universal primer: binds a variety of DNA templates.

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Description

Explore the various techniques of polymerase chain reaction (PCR) in this quiz. Understand the principles and applications of multiplex, nested, inverse, and reverse transcriptase PCR. Test your knowledge of these essential methods used in DNA amplification and analysis.