Lecture 9.1: Molecular Techniques and Diagnosis Flashcards
Molecular Techniques: Analysis of DNA/RNA
• PCR
• DNA/RNA electrophoresis
• Restriction analysis
• DNA sequencing
• Karyotyping
• FISH
• DNA Hybridisation
Molecular Techniques: Analysis of Proteins
• Protein electrophoresis
• Immunoassays
• Enzyme assays
• Mass spectrometry
Polymerase Chain Reaction (PCR)
A laboratory technique used to amplify DNA sequences based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand
Because DNA polymerase can add a nucleotide only onto a pre-existing 3’-OH group, it needs a primer to which it can add the first nucleotide
Components of PCR
- DNA template
- DNA primers
- dNTPs
- DNA polymerase (Taq* DNA polymerase)
- Buffer
Stages of PCR
1) Denaturation
2) Annealing
3) Elongation
Clinical applications of PCR
Allow amplification and analysis of DNA from a single cell, hair follicle, sperm for investigations in forensic medicine
The detection of viral, bacterial and protozoal agents
Diagnosis and genetic analysis of inherited diseases
Diagnosis and analysis of neoplastic disorders (including the detection of activated oncogenes)
Prenatal and pre-implantation diagnosis
Multiplex PCR
The multiplex polymerase chain reaction is a technique utilized for the amplification of multiple targets in a single PCR experiment
In a multiplex assay, more than one target sequence can be amplified using multiple primer pairs in a reaction mixture
DNA/RNA Gel Electrophoresis
A technique commonly used in laboratories to separate charged molecules like
DNA, RNA and proteins according to their size (smaller move further)
Charged molecules move through a gel, consisting of a permeable matrix, when an electric current is passed across it
Restriction Analysis
Based on Restriction Endonuclease (RE) enzymes produced by bacteria
REs recognise and cut a specific DNA sequence called restriction site
Used with DNA gel electrophoresis to investigate mutations, DNA variation and to clone DNA
Sanger DNA Sequencing
1) PCR fluorescent, chain-terminating ddNTPs (used to produce different lengths of DNA strands for DNA sequencing)
2) Size separation by capillary gel electrophoresis
3) Laser excitation & detection by sequencing machine
Next Generation Sequencing (NGS)
NGS aka massive parallel sequencing
Using NGS an entire human genome can be sequenced within a single day whilst Sanger required over a decade
1,000 Genomes Project
The goal of the 1000 Genomes Project was to find common genetic variants
with frequencies of at least 1% in the populations studied
10,000 Genomes Project (UK)
Studied nearly 10,000 individuals (4000 healthy and 6000 affected by disease)
Included rare inherited disorders and more common diseases such as autism, schizophrenia and obesity
Discovered contribution of rare variants, new genetic variants and genes underpinning disease risk
100,000 Genomes Project
Participants are NHS patients with a rare disease, plus their families, and patients with cancer
What is Karyotyping? Uses?
Karyotyping is the process of pairing and ordering all the chromosomes of an individual
Used to detect changes in chromosome number and also more subtle structural changes, such as chromosomal deletions, duplications, translocations, or inversions
Types of Karyotyping (2)
Giemsa Banding
Quinacrine Banding
Steps of FISH (Fluorescence In Situ Hybridisation)
Denaturing of DNA to separate strands to allow probe access to DNA
Hybridise together to bind probe to DNA
Analyse probe signals using fluorescence miscroscope
What is DNA Microarray Technology: “Genechip”?
A DNA microarray is a collection of synthetic DNA sequences (probes) attached to a spot, on a solid surface
How does DNA Microarray Technology: “Genechip” work?
Grid of DNA probes can hybridise to complementary “target” sequences derived from experimental samples to determine the expression level of specific mRNAs in a sample
Uses of DNA Microarray Technology: “Genechip”? (3)
To identify disease associated SNPs
To investigate chromosome deletions /duplications
To investigate conditional gene expression (i.e. tumour vs healthy tissue)
What is Protein Gel Electrophoresis?
SDS-PAGE is an analytical technique to separate proteins based on their molecular weight
Protein Gel Electrophoresis: Uses
• Estimate relative molecular mass
• Determine the relative abundance of major proteins in a sample
• Determine the distribution of proteins among fractions
• Asses the purity of protein samples
• Different staining methods can be used to detect rare proteins
• Specialised techniques can be used to detect extremely scarce gene products
What is an Immunoassay?
A biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes)
Monoclonal Antibodies
Produced by the same clone of plasma B cells
They bind to a single epitope
Epitope
The part of the antigen that binds to a specific antigen receptor on the surface of a B cell
Polyclonal Antibodies
Produced by different clones of plasma B cells
They bind to the many different epitopes in the same antigen
What is ELISA (Enzyme-Linked Immunosorbent Assay)?
A plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones
How does ELISA work?: Direct and Indirect
Direct: the antigen (target macromolecule) is immobilised on a solid surface (microplate) and then complexed with an antibody that is linked to a reporter enzyme
Indirect: Antigen in plate, blood/serum added, if Abs present they attach to antigen, then add secondary Abs with an attached enzyme, these secondary Abs will attach is primary Abs present, then add substrate to observe reaction
Uses of ELISA
Detection of platelet antibodies in serum used to identify patients diseases
ELISA was the first universal testing kit for HIV
ELISA is also frequently used in pregnancy test for the detection of hormone human chorionic gonadotrophin (hCG) in urine
Enzyme Assays
To measure the activity of clinically useful enzymes
The amount of enzymatic reaction product is measured
Common Enzyme Assays
Liver Function Tests (LFTs)
Mass Spectrometry
An analytical method used for measuring the mass-to-charge ratio (m/z) of one or more molecules present in a sample
