PCR Techniques Overview

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.