Molecular Methods Flashcards
What is molecular diagnositics
The use of molecular biology techniques to:
- Diagnose and monitor disease
- Detect current or future risk of disease
- Detect treatment guiding anomalies that will drive treatment of disease
What are molecular methods
Analysis of specific molecules e.g. Proteomics (proteins) or genomics (genes)
Mostly taken to be DNA and RNA analysis
DNA/RNA methods
- Amplification based - PCR
- hybridisation based - in situ hybridisation
What are the four methods of DNA analysis
PCR
Next generation sequencing
In situ hybridisation
Gene arrays
What are the two main methods of RNA analysis
PCR
In Situ hybridisation
What is in situ-hybridisation
Application of a labelled probe (oligonucleotide) to detect target DNA/RNA sequence of interest
Probe and a label
How does the probe for ISH work
Binds to target chromosome, gene or virus
How does the label work in ISH
Usually fluorescent
May be biotin or alkaline phosphatase
(FISH, CISH, SISH)
What is FISH
Fluorescent in situ hybridisation
What does CISH stand for
Chromogenic in situ hybridisation
What does SISH stand for
Silver in situ hybridisation
What are the five steps to the ISH methodology
- Pretreatment of cells (permeabilising)
- Apply probe, denature DNA
- Hybridise probe to target sequence
- Wash with buffers - stringency washes
- Detect signal through label
How is FISH carried out
Ratio of normal to test counted
e.g. Pink = Her2 and green = control chromosome 17
Ratio > 2.2 is amplified gene
Write about FISH gene amplification for HER2
Her2 gene amplification is assessed in breast and gastric tumours using FISH
20% of these have gene amplification of HER2 driving their tumour growth
If positive these can be treated with Herceptin
How is Her2 amplification treated
Herceptin drug
Its a monoclonal antibody called Trastzumab
It worked by blocking Her2 receptor and halting tumour growth
Write about the use of ISH for chromosomal translocation
In neoplasia part of one chromosome attaches to another incorrect chromosome
This leads to abnormal fusion product and mRNA followed by protein production
The chromosomal translocation may be used to diagnose or subtype neoplastic disease
Seen in:
- Leukaemias and lymphomas
- Sarcomas
What are the two types of translocations
Balanced
Unbalanced
What is a balanced translocation
There is even exchange of material with no genetic information extra or missing and is ideally fully functional
What is an unbalanced translocation
The exchange of chromosome material is unequal resulting in extra or missing genes
How is translocation EML4 ALK detectef
Uses a break apart probe
Occurs in some lung cancers
Yellow fluorescence = normal
Separated red and green signals = translocation
Write about PCR
Amplification of DNA and RNA
Creates millions of copies of a single sequence
What are four ways of analysing DNA and RNA
PCR
Gel electrophoresis
Sequencing
Arrays
Write about gel electrophoresis
Agar or polyacrylamide based gel
Visualise product from the PCR
Write about sequencing in the analysis of DNA/RNA
Sequencing of genes to establish exact sequence of product
Identification of a gene mutation
Write about arrays in DNA or RNA analysis
Evaluate DNA/RNA by binding it to fixed arrays of oligonucleotides
Identify multiple genetic changes simultaneously
What samples can undergo DNA/RNA analysis
Fresh frozen tissue - best method - good DNA/RNA quality
Paraffin-wax embedded tissue - DNA/RNA is fragmented, harder to extract for analysis
Cytology samples - DNA and RNA are easily released and analysed - easy to test for HPV this way
Write about DNA analysis by PCR amplification
A very specific method for amplifying a sequence of DNA or RNA so that it can be measured or sequenced
It is based on selection of primers which bind near the sequence of interest, so that it can be amplified
Any gene or exon can be amplified and analysed
This has revolutionised molecular biology, as it allows rapid analysis of DNA/RNA
What are the steps in PCR amplification
Most analysis is performed on nucleic acids extracted from cells/tissues
First step is harvesting and purifying this DNA/RNA
You must design your DNA primers
Denature your DNA
Anneal your primers
Extend your primers
Repeat
How can DNA be harvested and purified for PCR
Using proteolytic digestion of tissue to release DNA and/or RNA
DNA can then be purified (proteins removed etc) using Phenol/Chloroform, column or bead-based methods (spin columns with beads attached)
What chemicals are used to purify DNA
Phenol or chloroform
Give an example of a PCR analyser
Thermal cycler
Write about gel electrophoresis
May be used for multiplex PCR
Molecular weight marker (control) shows the relative sizes of the bands
Each band of the PCR reaction product - an Amplicon from the primers creating a product
Most labs use RTPCR machines so no gel electrophoresis is required
What is Real Time PCR
PCR with quantification/analysis of products
Tests and standards are used
Can be both DNA or RNA analysis
No gel electrophoresis needed instead you get a read out of the fluorescent results compared to standards
As gene product is replicated, fluorescent reporter signal is liberated
At each cycle a measurement is taken (real time)
Used in measuring a gene of interest vs a control gene
Increase or decrease in levels of a given varient
Write about Sanger sequencing
Gold standard of genomic testing
Amplified PCR products are subjected to a second round of PCR over shorter cycles and use a series of fluorescently tagged dideoxy nucleotides (ddNTP’s) which terminate chain extention
Product is cleaned and purified
Product is put through a polymer filled capillary tube and subjected to electrophoresis
The small pieces come through first and the sequence is read by the length of sequence
Write about next generation sequencing
Once PCR product is extracted it goes through library prep
This involved amplifying specific targets in the double stranded DNA
Digestion of the unbound primers. addition of a unique barcode (adapter), size selection and a clean up step
Each specimen is then quantified and equalised
Once equalised the samples are combined into one single tube
Samples are enriched and loaded on a chip
Chip is loaded onto a sequencer and information sent to bioinformatics platform
What are the two main applications of ISH and PCR in the cell path lab
Detection of microorganisms
Detection of tumour-associated genetic alterations
What microrganisms are detected via molecular methods
Human papillomavirus - for cytology/cervical screening
Epstein-Barr virus - for lymphomas
Human Herpes virus 8 - Kaposi sarcoma
How can ISH and PCR be used to detect tumour associated genetic alterations
Mutations
Chromosomal translocations
Gene expression (amplification, loss, hypermethylation)
What is the most used real time PCR system
Roche Cobas 4800 system HPV
Write about the Roche Cobas 4800 system HPV
Real time PCR analyser
94 samples
Automated sample preparation
Rapid turnaround time
FDA approved, pooled PCR test for high risk and specific test for HPV
Write about gene mutation analysis
A new area of development for cancer diagnostics and therapy
Mutations with cancer predisposition are well known
- Inherited e.g. BRCA 1 and 2, APC, p53
- Somatic - BRAF, KRAS, EGFR
Mutations in cancer cells may be predictive of tumour progression and therapy response or resistance
Write about KRAS mutation
KRAS mutation in colon cancer
Anti-EGFR therapy will not work if positive for this
Write about EGFR mutation in lung
Tumour will be sensitive to anti-EGFR therapy if positive
Write about BRAF mutation
Seen in melanoma
If mutation present the tumour may be sensitive to b-raf tyrosine kinase inhibitor therapy
Write about circulating tumour cells
(5)
Tumour cell that have shed into the vasculature or lymphatics
No need for a biopsy as can take a blood sample
Rapid turnaround of results
Mainly used in the detection of resistance mutations
Still in development for most biomarkers
