PATH3305 L5a & L5b: Detection of Genetic Variation Techniques and Applications

Mutation Detection/Genotyping

Refers to the determination or identification of specific genetic (DNA) variations.

Applications of Mutation Detection/Genotyping

Research: Identifying disease-related genes. Diagnostics: Confirming mutations, assessing prognosis. Pharmacogenomics: Predicting treatment response.

Mutations

May arise de novo, be tissue-restricted, and occur independently in the same gene across individuals.

Polymorphisms

Stable, germline variations present in all tissues and shared at the same genomic position across individuals.

Known unknowns

Mutations.

Known knowns

Polymorphisms.

Unknown unknowns

Undiscovered variants.

Genotyping

Determines the actual DNA sequence and identifies specific genetic variations. Allows prediction of phenotype.

Phenotyping

Observes physical or biochemical traits, infers underlying genotype.

Variant affect on proteins and phenotype

Most variants do not change protein properties or phenotype.

Genotyping Accuracy

Genotyping is more accurate when the variant does not cause observable phenotypic changes.

General Workflow of Genotyping

Decision to test. Sample collection (e.g. blood, tissue). Genotyping method selection (controls, accuracy). Interpretation (e.g. homozygous/heterozygous).

Basis of PCR Amplification

Rapid, specific amplification using primers.

What is Polymerase Chain Reaction (PCR)

A molecular biology technique used to amplify a specific DNA sequence exponentially in vitro.

Uses of PCR

Pathogen detection (e.g. viral, bacterial DNA), Mutation analysis, Genotyping, Cloning and gene expression studies.

PCR Components

Template DNA (what you want to copy), Primers (short sequences that bind to the target region), DNA polymerase (an enzyme that builds new DNA), Nucleotides (DNA building blocks).

Principle of PCR

PCR relies on thermal cycling and sequence-specific primers to target and amplify DNA using a thermostable DNA polymerase (e.g. Taq polymerase).

PCR Steps

Denaturation (∼95°C): Double-stranded DNA is separated. Annealing (∼50-65°C): Primers hybridise to complementary target sequences. Extension (∼72°C): DNA polymerase synthesises new DNA from primers.

Exponential Amplification in PCR

Each cycle doubles the amount of target DNA, resulting in exponential amplification.

Restriction Enzyme Digestion

Cuts DNA at specific sequences (palindromes).

Separation Methods

Electrophoresis: Agarose: good for large fragments. Acrylamide (PAGE): better resolution for smaller fragments.

Detection Approaches

Ethidium bromide (general). Fluorescent or radioactive probes (specific).

Sanger Sequencing

Chain-terminating nucleotides (ddNTPs) stop elongation during PCR; fragments are separated by size via capillary electrophoresis.

SNVs

Small variants in a known gene/region.

Small indels

Insertions or deletions of 1-20 base pairs.

ssDNA

Single-stranded DNA.

dsDNA

Double-stranded DNA.

qPCR

Amplifies DNA using specific primers; fluorescence emitted is proportional to amount of product formed.

Known variants

Includes known SNVs, CNVs, RNA expression, presence or absence of a gene.

cDNA

Complementary DNA synthesized from RNA.

MLPA

Probes hybridise to adjacent target sites, ligated only if both bind, followed by PCR and capillary electrophoresis.

CNVs

Copy number variations, including deletions or duplications of ≥ 1 exon.

Microarray

Target DNA/RNA hybridises to probes on a chip; fluorescent signal indicates hybridisation strength.

FISH

Fluorescent probes bind to specific chromosome regions; visualised under fluorescence microscopy.

Karyotyping

Stains and spreads metaphase chromosomes to visualise number and structure under a microscope.

Massively Parallel Sequencing

DNA is fragmented and sequenced in parallel; data aligned to a reference genome for variant calling.

Clinical testing

Purpose of testing for clinical applications.

Research testing

Purpose of testing for research applications.

Diagnostic testing

Purpose of testing for diagnostic applications.

Screening

Purpose of testing for screening applications.

Data integrity

Multiple handling steps increase contamination risk.

Pros of Fluorescent-Based Detection

Closed-tube, low contamination risk, efficient and quick, suitable for high-throughput testing.

Cons of Fluorescent-Based Detection

High cost of probes and instrumentation; requires technical expertise.

MALDI-TOF MS

Uses mass difference to detect SNPs.

Thermal Melting Curve

Detects changes in DNA binding and melting behaviour.

SSCP

Single-strand DNA conformational changes detected via electrophoresis.

Steps of Sanger Sequencing

Denature DNA to single strands, anneal a primer near the 3' end of the sequence of interest, set up four reactions each with DNA template, primer, DNA polymerase, normal dNTPs, and a small amount of one labelled ddNTP.

Electrophoresis in Sanger Sequencing

Electrophoresis (polyacrylamide gel) separates products by size, allowing the sequence to be read based on fluorescent or radiolabelled ddNTPs at the end of each fragment.

Advantages of Sanger Sequencing

High accuracy and is the gold standard for confirming variants.

Limitations of Sanger Sequencing

Low throughput, time-consuming, expensive, and limited by the number of reactions and capillaries.

Sanger Sequencing vs Pyrosequencing - Mechanism

Sanger Sequencing uses chain termination while Pyrosequencing uses sequencing by synthesis.

Sanger Sequencing vs Pyrosequencing - Detection

Sanger Sequencing detects using fluorescent ddNTPs and electrophoresis, while Pyrosequencing detects light emission via luciferase.

Sanger Sequencing vs Pyrosequencing - Reaction Complexity

Sanger Sequencing is relatively simple, whereas Pyrosequencing involves a complex enzyme system.

Sanger Sequencing vs Pyrosequencing - Throughput

Sanger Sequencing has low throughput compared to Pyrosequencing which has low to medium throughput.

Sanger Sequencing vs Pyrosequencing - Cost

Sanger Sequencing has moderate to high cost, while Pyrosequencing is high.

Key Features of Next-Generation Sequencing (NGS)

Massively parallel sequencing of millions of fragments, eliminates need for electrophoresis.

General Workflow of NGS

Fragment DNA and ligate universal adaptors, amplify DNA fragments on beads or surface (e.g. bridge PCR), sequence each clonal amplicon (1 base at a time), assemble reads using reference-based or de novo methods.

Illumina Platforms

Includes HiSeq, MiSeq, NextSeq, NovaSeq, and uses sequencing by synthesis.

SOLiD Platform

Developed by Applied Biosystems, it uses ligation-based sequencing.

Ion Torrent Platform

Detects pH changes, no optics, includes PGM/Proton.

Roche 454/FLX Platform

Based on pyrosequencing.

Pacific Biosciences Platform

Uses SMRT/Sequel for single molecule real-time sequencing.

Oxford Nanopore Platform

Includes MinION/GridION, nanopore-based, no synthesis or labelling.

Illumina (SBS) - Read Length

Short.

Illumina (SBS) - Throughput

Very high.

Ion Torrent - Use Case

Fast diagnostics and small genomes.

Whole-exome sequencing

Identifies coding region mutations.

Whole-genome sequencing

Detects structural variants, SNPs, and CNVs.

Targeted panels

Focus on known disease genes (e.g. cancer genes).

Transcriptome (RNA-seq)

Analyses gene expression changes.

ABI 3730XL - Throughput

Low, 50 kb.

Roche GS-FLX - Cost per Mb

$25-30 (raw).

Illumina HiSeq - Output

500-600 Gb.

Nanopore Sequencing

Directly senses DNA as it passes through a nanopore, measures changes in current, and promises portability, real-time data, and long read lengths.