MLPA

Question 1

What proportion of beta thalassaemia is caused by a deletion?

Answer 1

Approx 10%. Therefore need to sequence gene first before doing MLPA

Question 2

What proportion of alpha thalassaemia is caused by a deletion?

Answer 2

Approx 90%

Question 3

Why is RNAse added for alpha/beta MLPA?

Answer 3

Hb is highly expressed so mRNA can bind to probes which reduces the concentration of the probes

Question 4

Why/when is molecular testing done for thalassaemia?

Answer 4

1. Prenatal screening / diagnosis
2. To diagnose a thalassaemia not diagnosed by other methods

Question 5

What probes in the alpha thal MLPA kit detect a sequence common to both the a1 and a2 genes?

Answer 5

172, 214, 220 nt probes

Question 6

What are 6 limitations of MLPA alpha thal testing?

Answer 6

1. False positives - SNPs, mutations or indels under probe
2. False negatives - may be caused by deletion and duplication of similar size on different chromosomes
3. Non-deletional alpha thal not detected by MLPA (except for HbCS)
4. Some deletions can’t be differentiated between e.g –FIL and –THAI (need to do PCR)
5. Unable to determine if some deletions (those that don’t overlap) are in cis or trans
6. Can’t determine zygosity of Hb CS ==> need to sequence gene

Question 7

When is MLPA recommended for beta thalassaemia?

Answer 7

When beta thalassaemia is suspected but sequence analysis of the beta-globin gene is negative

Question 8

How would you design an MLPA probe?

Answer 8

1. Probe consists of LHS, RHS, primer binding sequences and sometimes a stuffer sequence
2. Is the probe being designed to detect CN change or a mutation?
3. General probe design rules:
   - LHS and RHS should be adjacent for the ligase enzyme to work
   - Each probe in multiplex reaction should have a different length and should have unique hybridising sequences i.e should not be overlapping
   - Minimum length of LHS or RHS: 21 nucleotides, preferably 26-30 nucleotides.
   - Tm of each hybridising sequence (LHS/RHS separately) should be ≥ 68°C (preferably ≥ 71°C) n.b The advantage of a long hybridising sequence and a high Tm is that the probe signal will be less sensitive to polymorphisms (SNPs) within the hybridising sequences.
   - GC content should be ~ 50%
   - Avoid homopolymers
   - The ligase-65 enzyme is most sensitive to mismatches at the 3’ end of the LPO (LPO side of the ligation site).
4. Design probe hybridising sequence and check against tool such as BLAST NCBI to ensure uniqueness and not including SNPs

Question 9

What are the key steps of an MLPA assay (7)?

Answer 9

1. Denature sample
2. Add master mix of probes and MLPA buffer and allow to incubate at 60 degrees for 16-20 hours (for hybridisation)
3. Add ligase to ligate the LHS and RHS
4. Add universal primers and PCR mix N.B. the essence of MLPA is that it is not the sample DNA that is amplified, but the MLPA probes that are added to the sample
5. The forward PCR primer is fluorescently labelled to facilitate detection at the next step.
6. Separate PCR products using capillary electrophoresis
7. Determine the relative copy numbers in the sample