20.02.01 Mutation scanning

Question 1

What is mutation scanning

Answer 1

Searching for novel sequence variants within a define DNA fragment

Question 2

Factors that influence the choice of scanning method used

Answer 2

• Detection sensitivity
• Suitability according to sample type (peripheral blood or tumour tissue)
• Suitability for predicted mutation type (PTT for polypeptide chain terminating mutations)
• Features of the DNA sequence analysed (presence of polymorphisms)
• Healthy and safety (toxic chemicals required)
• Sample throughput
• Equipment and running costs
• Post PCR manipulation requirements (lots of post PCR steps)

Question 3

What is DHPLC

Answer 3

• Denaturing high performance liquid chromatography
• Relies on the creation of hetero- or homo-duplexes of paired DNA in order to identify SNPs.
• Products are then separated using a denaturing liquid chromatography column, which denatures the duplexes

Question 4

What is MALDI-TOF

Answer 4

• Matrix-assisted laser desorption/ionisation time of flight mass spectrometry
• Determines the mass of a molecule by measuring the mass-to-charge ratio (m/Z) of it’s ions.

Question 5

Steps of MALDI-TOF

Answer 5

1. Sample is placed on a UV absorbing matrix pad and exposed to a laser pulse
2. Ionised sample is accelerated and moved in an electric field towards detector
3. Time of flight required to reach the detector depends on the m/z of individual molecules.

Question 6

Uses of MALDI-TOF

Answer 6

• Used to measure DNA molecules up to 20kD
• Can size oligonucleotides 100bp faster than electrophoretic separation
• Can determine base composition
• Can analyse proteins transcribed from DNA to detect sequence variation

Question 7

Benefits of MALDI-TOF

Answer 7

-Efficient ionisation of DNA molecules
-Accurate determination of mass
-Faster
-High throughput
Allele frequencies of a point mutation can be estimated in analysis of a pooled sample

Question 8

Example of MALDI-TOF

Answer 8

• Sequenom iPLEX kit

- Contains mass-modified dideoxynucleotide terminators

Question 9

Disadvantages of MALDI-TOF

Answer 9

• Expensive

- Large bits of specialist equipment required

Question 10

What is HRM

Answer 10

• High resolution melt curve analysis

- Detects differences in sequence based on an altered melting temperature (Tm)

Question 11

Steps in HRM

Answer 11

1. PCR-based amplification of region of interest in presence of dsDNA binding dye which is fluorescent only when bound to dsDNA.
2. Fluorescence is measured whilst amplicon is subjected to gradual increase in temperature. When Tm is reached DNA duplex separates and dye is released, fluorescence therefore decreases.
3. Plot fluorescence vs temperature.

Question 12

What intercalating dyes should be used in HRM

Answer 12

• Ones that don’t preferentially bind to specific bases, that do not inhibit PCR and do not influence Tm.
• SYBR green- unsuitable as inhibits polymerase and stabilises duplex
• SYT09. Doesn’t alter reaction dynamics even at high concentrations.
• Evagreen. Fluorescent signal is quenched when in solution. Quencher is released on binding.

Question 13

What factors would affect HRM

Answer 13

• Extraction method. All DNAs being analysed should be extracted using same methodology
• DNA concentration. At low template concentration there is a greater chance of incorporating a mutation early in the PCR which will affect the melting behaviour. At high concentration there will be high background fluorescence due to increased intercalation of dye into dsDNA
• Higher concentration of MgCl2 increases number of mispaired heterozygote samples

Question 14

Applications of HRM

Answer 14

Genotyping, mutation scanning and methylation analysis (bisulphite modification prior to PCR)

Question 15

Advantages of HRM

Answer 15

• Fast
• Closed tube system, limits contamination
• Cheap (only high cost is the machine)
• High throughput
• PCR products can be used for other applications (e.g. sequencing if aberrant melt profile)

Question 16

Disadvantages of HRM

Answer 16

• Not 100% sensitive
• Identifies presence of variant but does not characterise it (further work required, sequencing)
• Not suitable for highly polymorphic genes
• Need small fragments (50-150bp), with no more than 1-2 melt domains.
• Needs extensive optimisation
• Better for analysing large numbers of samples to enable accurate data analysis.

Question 17

What happens in HRM

Answer 17

• Following PCR there will be a pool of 4 different amplicons (2 het duplexes and 2 hom duplexes). V/W, W/V, V/V, W/W
• 4 different melt profiles in one fluorescence signal output.
• hets are easily identified as a double peak. Due to more unstable heteroduplex pairing, they will melt at a relatively lower temperature.

Question 18

What is PTT

Answer 18

• Protein truncation test
• Detects mutations that create premature termination codons (nonsense, splice, frameshift)
• Special primers that contain RNA pol promoter sequence and eukaryotic initiation sequences to allow in vitro transcription and translation

Question 19

Steps in PTT

Answer 19

• Amplification of region using PCR/ RT-PCR
• In vitro transcription/ translation of region
• SDS-PAGE for protein analysis. Shorter protein products from mutated alleles are easily distinguished from wild type.

Question 20

Advantages of PTT

Answer 20

• Suited to genes where majority of mutations are truncating. BRCA1/2, APC
• Large coding regions can be covered in one fragment
• Can determine approximate location of mutation
• Sensitivity of (5-10%)

Question 21

Disadvantages of PTT

Answer 21

• Time consuming and outdated
• Use of radiolabels.
• Missense variants not detected.
• Deletions spanning segment being analysed or 5’ end will be missed
• Low throughput.

Question 22

What is enzymic cleavage of heteroduplexes

Answer 22

-Use of endonucleases to cleave dsDNA at heteroduplexes

Question 23

Steps in enzymic cleavage of heteroduplexes

Answer 23

• Amplification of region of interest using PCR or RT-PCR
• Digest with enzyme
• Gel electrophoresis

Question 24

Advantages of enzymic cleavage of heteroduplexes

Answer 24

• Rapid, cheap
• Can analyse long range PCR products that are difficult to sequence
• Can detect multiple variants in a single PCR product
• Could be automated

Question 25

Disadvantages of enzymic cleavage of heteroduplexes

Answer 25

- Detection rate is 92-95% | - Still need to sequence to characterise mutations