Lecture 16 Part 2 - MLPA

Question 1

What is MLPA

Answer 1

multiplex ligation-dependent probe amplification (proprietary technique)

Question 2

What are the steps of MLPA

Answer 2

1. denaturation
2. probe hybridixation
3. ligation
4. PCR
5. capillary electrophoresis
6. analysis

Question 3

What are the parts of an MLPA probe

Answer 3

primer, target specific region, stuffer sequence

Question 4

What happens if the primers don’t ligate

Answer 4

the PCR primer binding site does not exist, and it cannot replicate

Question 5

What is QC for MLPA

Answer 5

• benchmark fragment in the master mix
• quality fragments to ensure the correct amount of sample DNA has been added
• D fragments that ensure denaturation was complete
• X and Y fragments to identify sample mix ups
• 3 reference samples from healthy individuals
• positive control from individuals with known copy number variants

Question 6

How to read MLPA

Answer 6

Reference has 2 copies, patient could have 1x, 0.5x or 1.5x