Low-Level Mutation Detection Flashcards
1
Q
What is low level mutation detection?
A
- Detection of a variant population of DNA in a sample where the wild-type (wt) DNA greatly exceeds the variant DNA contribution.
- Need high selectivity and enrichment for successful detection and identification.
2
Q
What are the key applications of low level mutation detection?
A
- Somatic muts in tumour samples which are a heterogeneous mix of wt & tumour DNA (tumour itself is a heterogeneous mix of cells reducing mut level further.
- Muts in fetus using NIPD, in which the cell-free fetal DNA load is only 3-6% of the entire DNA population (i.e. maternal DNA).
- Heteroplasmic muts in mtDNA genomes, where mutant mtDNA is a proportion of wt mtDNA
3
Q
What is mutation ‘enrichment’ vs mutation ‘selectivity’?
A
- Selectivity of a mutation detection method refers to the selection of mutation-containing alleles among an excess of wild-type alleles
- Enrichment refers to a process that increases mutant allele concentration relative to wild-type alleles.
- The use of enrichment methods and highly selective assays is often necessary to facilitate low-level mutation detection.
4
Q
What are the different classes of mutation ‘enrichment’ methodology?
A
- Methods differ by their ability to enrich either known or unknown muts.
- Design of mut enrichment assays for the detection of known muts is much easier than for unknown muts as specific nucleotides can be targeted
- As a result more methodologies exist for enrichment of known muts than unknown muts.
5
Q
What different strategies can be used to enrich and/or select for low-level mutations when the mutation is known?
A
- Preferentially destroying/blocking the wt allele
- Utilised restriction enzymes
- Preferentially amplifying the variant allele
- Utilises allele specific primer sequences
- Spatially separating the variant from the wt allele
- Utilises specialised plates with micro-chambers or lipid droplets in emulsion
6
Q
Name various methods that use preferentially destroying/blocking of the wt allele (moderate selectivity).
A
- Restriction Endonuclease-Mediated Selective PCR (REMS-PCR)
- Variant alters the sequence of a restriction enzyme (RE) site, inclusion of that RE during PCR will cause digestion of the wt amplicons (with the intact RE site) and amplification products only for the mutated allele (mutated RE site > not recognised by RE > PCR product).
- Artificial Introduction of a Restriction Site (AIRS) RFLP
- If the mut does not alter a RE site, AIRS uses a modified primer that selectively binds to the wt allele and introduces a RE site into the wt PCR product during PCR > wt product of a smaller size > fragments separated by gel electrophoresis.
- Peptide Nucleic Acid (PNA)-mediated PCR and Locked Nucleic Acid (LNA)-mediated PCR
- Under appropriate PCR cycling conditions, the chemically modified PNA or LNA probes specific for the wt allele will bind to the wtDNA and block primer annealing, facilitating amplification of only the variant molecules.
7
Q
Name various methods that use preferentially amplifying of the variant allele (moderate selectivity).
A
- AS-PCR (Allele-Specific PCR; key technique),
- ARMS (Amplification Refractory Mut System),
- PASA (PCR Amplification of Specific Alleles),
- PAMSA (PCR Amp of Multiple Specific Alleles),
- TaqMan or Scorpian Real-time PCR
8
Q
Name a method that has very high selectivity and exploits spatially separating the variant from the wt allele.
A
Digital PCR;
- Within a sample individual nucleic acid molecules are partitioned into separate regions.
- Each partition contains either a negative or positive reaction, i.e. “0” or “1” (i.e. digital output).
- These regions can be generated using, micro well plates, capillaries, emulsion PCR and nanofluidic chips.
- This separation allows absolute quantification (without the need for a standard curve) and a more reliable collection and sensitive measurement of nucleic acid amounts.
9
Q
Name another method with high selectivity for detection of known mutations
A
MALDI-TOF MS
10
Q
What are the two main methods of enriching/selecting for unknown mutations?
A
- COLD-PCR (Co-amplification at lower denaturation temperature PCR) followed by a downstream detection method e.g. Sanger sequencing, MALDI-TOF
- NGS
11
Q
What is COLD-PCR?
A
COLD_PCR is protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA.
12
Q
What is the principle of COLD-PCR?
A
- Single nucleotide mismatches will slightly alter the melting temperature (Tm) of the double-stranded DNA (Tm changes of 0.2-1.5 °C are common)
- Each dsDNA has a ‘critical temperature’ (Tc) lower than its Tm. The PCR amplification efficiency drops measurably below the Tc.
- Set an intermediate annealing temperature that allows hybridization of mutant and wildtype heteroduplexes
- Heteroduplexes are selectively denatured at the Tc
- The homo-duplex DNA will preferentially remain double stranded at the Tc and not be available for primer annealing - thus is not amplified in the extension stage.
13
Q
What are the variations of COLD-PCR?
A
- Full COLD-PCR – Enrichment of all possible muts.
- Induces the formation of heteroduplexes at positions of muts.
- By using a lower denaturation temperature during PCR, double-stranded DNA (dsDNA) containing mismatches (heteroduplexes) denature first.
- True homoduplexes have a higher melting temperature (Tm) & denature less than heteroduplexes at the Tc; thus their amplification is relatively suppressed.
- Fast COLD-PCR – Enrichment of known Tm-reducing muts (vs. wt).
- Selectively denature only variant sequences by denaturing at the Tc and then only amplify these
14
Q
What are the advantages of using COLD-PCR?
A
- Single-step method capable of enriching both known and unknown minority alleles irrespective of mutation type and position.
- Does not require any extra reagents or specialized machinery.
- Therefore the cost is not increased. Better than conventional PCR for the detection of mutations in a mixed sample. Does not significantly increase experiment run time compared to conventional PCR.
15
Q
What are the disadvantages of using COLD-PCR?
A
- Vulnerable to polymerase induced error, needs sequences less than 200bp
- All variants may not have a Tc that differs significantly from the wt
- Success depends on the sequence and position of the variant as this affects the dynamics of amplification.
16
Q
What pre-conditions are required is one is to utsilise NGS to detect low-level mutations?
A
- In order to detect low level muts by NGS, much higher read depth (deep sequencing) is needed.
- To detect rare single nucleotide variants at 0.1% requires robust processing methods, data pipeline, 10,000’s high quality parallel reads (see: Cushing et al 2013).
- Beyond this level (<0.1%) it begins to become impractical (unit cost, PCR resampling, data volume) to use NGS read depth alone to detected minority populations.
