Types of PCR: Multi-plex PCR

Multi-plex PCR

• More than one primer pair can be included in a single PCR reaction.
• Enables simultaneous amplification of multiple DNA products.
• Particularly useful for typing or identification analyses.
• Requires complex optimization of reagents and conditions.
• Target sequences may amplify at different efficiencies, posing challenges.
• Potential primer interference can affect binding to target sequences.
• PCR conditions must be carefully adjusted to ensure optimal amplification.

Reverse Transcriptase PCR (RT-PCR)

• Measures gene expression by converting RNA to cDNA before amplification.
• cDNA synthesis is catalyzed by reverse transcriptase, utilizing mRNA as a template.
• mRNA is fragile and cannot be amplified directly by PCR.
• Reverse transcriptase was first discovered in retroviruses, which have RNA genomes.
• The RT-PCR process includes:
  • Binding an oligo(T) primer to the 3’ poly(A) tail of mRNA.
  • Synthesizing cDNA strands from RNA templates.
  • Separating RNA-cDNA hybrids through temperature increase.
  • Using gene-specific primers for further amplification.
• Final amplification results in billions of copies of the expressed gene.
• The quantity of DNA is visualized using gel electrophoresis.

Nested PCR

• Enhances sensitivity essential in clinical applications where specimen quality is often compromised.
• Addresses challenges posed by low target abundance and interfering sequences.
• Increases reliability of PCR outcomes in clinical diagnostics.

Real-Time (Quantitative) PCR (qPCR)

• Measures PCR product amounts at each amplification cycle in real-time.
• Uses fluorescent dyes for detection during or after the amplification process.
• Examples include SYBR Green and ethidium bromide (EtBr) for dye-based detection.
• Detection methods:
  • DNA intercalating or minor groove-binding dyes that fluoresce more as template DNA increases.
  • Sequence-specific DNA probes labeled with fluorophores that rely on Taq polymerase’s exonuclease activity.
• The polymerase separates fluorophores from quencher molecules, leading to increased fluorescence.
• The threshold cycle (CT) indicates the PCR cycle number at which fluorescence crosses a pre-defined threshold.
• Fluorescence accumulation is represented as a sigmoid curve over multiple cycles, with earlier fluorescence indicating more starting template.

Positional Cloning

• Laboratory technique used to locate disease-associated genes on chromosomes.
• Functions without prior knowledge of the gene's protein role in the disease.
• Utilizes known polymorphic markers to trace inheritance patterns in affected families.

Steps in Positional Cloning

• Begins with genetic analysis of patients and their families to define critical DNA regions.
• Statistical estimation determines if disease traits segregate independently or are linked to specific molecular markers.
• Identification of markers linked to disease manifestations.

Restriction Fragment Length Polymorphism (RFLP)

• Technique developed by Alec Jeffreys in 1984 for studying hereditary diseases.
• Analyzes unique patterns in DNA fragments through Variable Number of Tandem Repeats (VNTRs).
• Genetic polymorphism refers to inherited genetic differences in over 1% of the population.
• RFLP exploits DNA sequence differences for genetic differentiation (intraspecies and interspecies).
• Markers are typically co-dominant and highly locus-specific, allowing detection of both alleles in heterozygous samples.

RFLP Probes

• Labeled DNA sequences that hybridize with digested DNA fragments separated via gel electrophoresis.
• Reveal unique blotting patterns for specific genotypes at designated loci.
• Used for genome mapping, genetic variation analysis, forensics, paternity testing, and hereditary disease diagnosis.

Application: Sickle Cell Anemia Detection by RFLP

• A mutation causes the alteration of restriction sites within the gene or flanking regions.
• Sickle-cell anemia involves a single base-pair change resulting in Hb-S instead of normal Hb-A.
• The mutation modifies the sixth codon of β-globin from GAG (glutamic acid) to GUG (valine), affecting hemoglobin structure.
• Abnormal aggregation of Hb-S leads to the characteristic sickling of red blood cells.