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2.2 cellular pathology > Molecular Methods > Flashcards

Molecular Methods Flashcards

1
Q

What is molecular diagnositics

A

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

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2
Q

What are molecular methods

A

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

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3
Q

What are the four methods of DNA analysis

A

PCR
Next generation sequencing
In situ hybridisation
Gene arrays

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4
Q

What are the two main methods of RNA analysis

A

PCR
In Situ hybridisation

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5
Q

What is in situ-hybridisation

A

Application of a labelled probe (oligonucleotide) to detect target DNA/RNA sequence of interest

Probe and a label

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6
Q

How does the probe for ISH work

A

Binds to target chromosome, gene or virus

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7
Q

How does the label work in ISH

A

Usually fluorescent

May be biotin or alkaline phosphatase

(FISH, CISH, SISH)

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8
Q

What is FISH

A

Fluorescent in situ hybridisation

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9
Q

What does CISH stand for

A

Chromogenic in situ hybridisation

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10
Q

What does SISH stand for

A

Silver in situ hybridisation

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11
Q

What are the five steps to the ISH methodology

A
  1. Pretreatment of cells (permeabilising)
  2. Apply probe, denature DNA
  3. Hybridise probe to target sequence
  4. Wash with buffers - stringency washes
  5. Detect signal through label
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12
Q

How is FISH carried out

A

Ratio of normal to test counted
e.g. Pink = Her2 and green = control chromosome 17

Ratio > 2.2 is amplified gene

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13
Q

Write about FISH gene amplification for HER2

A

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

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14
Q

How is Her2 amplification treated

A

Herceptin drug

Its a monoclonal antibody called Trastzumab

It worked by blocking Her2 receptor and halting tumour growth

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15
Q

Write about the use of ISH for chromosomal translocation

A

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

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16
Q

What are the two types of translocations

A

Balanced
Unbalanced

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17
Q

What is a balanced translocation

A

There is even exchange of material with no genetic information extra or missing and is ideally fully functional

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18
Q

What is an unbalanced translocation

A

The exchange of chromosome material is unequal resulting in extra or missing genes

19
Q

How is translocation EML4 ALK detectef

A

Uses a break apart probe
Occurs in some lung cancers
Yellow fluorescence = normal
Separated red and green signals = translocation

20
Q

Write about PCR

A

Amplification of DNA and RNA
Creates millions of copies of a single sequence

21
Q

What are four ways of analysing DNA and RNA

A

PCR
Gel electrophoresis
Sequencing
Arrays

22
Q

Write about gel electrophoresis

A

Agar or polyacrylamide based gel
Visualise product from the PCR

23
Q

Write about sequencing in the analysis of DNA/RNA

A

Sequencing of genes to establish exact sequence of product
Identification of a gene mutation

24
Q

Write about arrays in DNA or RNA analysis

A

Evaluate DNA/RNA by binding it to fixed arrays of oligonucleotides

Identify multiple genetic changes simultaneously

25
Q

What samples can undergo DNA/RNA analysis

A

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

26
Q

Write about DNA analysis by PCR amplification

A

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

27
Q

What are the steps in PCR amplification

A

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

28
Q

How can DNA be harvested and purified for PCR

A

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)

29
Q

What chemicals are used to purify DNA

A

Phenol or chloroform

30
Q

Give an example of a PCR analyser

A

Thermal cycler

31
Q

Write about gel electrophoresis

A

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

32
Q

What is Real Time PCR

A

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

33
Q

Write about Sanger sequencing

A

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

34
Q

Write about next generation sequencing

A

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

35
Q

What are the two main applications of ISH and PCR in the cell path lab

A

Detection of microorganisms

Detection of tumour-associated genetic alterations

36
Q

What microrganisms are detected via molecular methods

A

Human papillomavirus - for cytology/cervical screening
Epstein-Barr virus - for lymphomas
Human Herpes virus 8 - Kaposi sarcoma

37
Q

How can ISH and PCR be used to detect tumour associated genetic alterations

A

Mutations
Chromosomal translocations
Gene expression (amplification, loss, hypermethylation)

38
Q

What is the most used real time PCR system

A

Roche Cobas 4800 system HPV

39
Q

Write about the Roche Cobas 4800 system HPV

A

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

40
Q

Write about gene mutation analysis

A

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

41
Q

Write about KRAS mutation

A

KRAS mutation in colon cancer

Anti-EGFR therapy will not work if positive for this

42
Q

Write about EGFR mutation in lung

A

Tumour will be sensitive to anti-EGFR therapy if positive

43
Q

Write about BRAF mutation

A

Seen in melanoma

If mutation present the tumour may be sensitive to b-raf tyrosine kinase inhibitor therapy

44
Q

Write about circulating tumour cells
(5)

A

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

2.2 cellular pathology (29 decks)

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