MLPA

Question 1

Define MLPA

Answer 1

Multiplex Ligation-Dependent Probe Amplification:
- for detection of deletions, duplications and aneuploidies

Question 2

Describe MLPA process (6)

Answer 2

1. Denaturation
2. Probe hybridization
3. Ligation
4. PCR
5. Capillary electrophoresis
6. Analysis

Question 3

Describe Probe design in MLPA

Answer 3

Left probe: codes Primer 2 site + target
Right probe: Stuffer + Primer 1 site

Stuffer region = (random DNA) adjusts length of product (ie. better resolution)

Question 4

Describe Ligation MLPA

Answer 4

Both probe sequences must match target DNA = close proximity = ligation

Question 5

Describe Amplification in MLPA

Answer 5

• amplifies ligated probe and subsequent amplicons simultaneously

Cycle 1:
- denature ligated probe from DNA template
- primer 1 binds to Right probe
- extension from ligated probe

Cycle 2:
- denature amplicon from ligated probe
- labeled primer 2 binds on the Left of amplicon
- extension from amplicon

Question 6

How are amplicons detected in MLPA ?

Answer 6

Capillary electropheroesis:
- Each sample gets a DNA size standards
- denaturation = ss
- detects labeled ss amplicons (from Primer 2)

Question 7

ID Quality control fragments specific for MLPA (4)

Answer 7

1. Benchmark fragment
   - ensures fragments are detected properly
2. Quantity (Q) fragments:
   - ensures correct amount of sample DNA was added
3. D fragments:
   - ensures denaturation was complete
4. X and Y fragments:
   - can identify sample mix-ups

Question 8

Other Quality control (3) in MLPA

Answer 8

1. No Template DNA control
   - only 4 Quality fragments should be visible
2. Three reference/ negative controls (DNA from healthy individuals with a normal copy number)
3. Positive control DNA from individuals with known copy number variants

Question 9

T or F: MLPA probes are very specific and can detect point mutations

Answer 9

TRUE; MLPA probes are very specific and can detect point mutations

Question 10

What happens when the two probes are not ligated in MLPA ?

Answer 10

• both probes did not match DNA sequence specifically = no ligation
• ONLY RIGHT PROBE IS AMPLIFIED

Cycle 1:
- denature probes from target DNA
- primer 1 binds Right probe
- extension of Right probe ONLY

Cycle 2:
- denature amplicon from Right probe
- labeled primer 2 cannot bind; Left probe was not amplified

Question 11

How is capillary electrophoresis after MLPA used to detect the gain or loss of a chromosome ?

Answer 11

• Ratio of Fluorescence from labeled amplicons
• Compare patient to control