MLPA

Question 1

MLPA

Answer 1

Multiplex Ligation-Dependent Probe Amplification:

• ## detection of deletions, duplication,s 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

Probe hybridization design

Answer 3

• 3’ end hybridizes to template
• 5’ end: binds

Green: template specific
Yellow: constant for all primers
Grey: stuffer region (random DNA) adjusts length of product (ie. better resolution)

Question 4

Ligation

Answer 4

Two probes must be in close prioximity
- sequences must match

Question 5

WIP PCR

Answer 5

• amplifies ligated probe

Cycle 1:
- denature template from probe
- primer 1 binds to stuffer leg
- extend

Cycle 2:
- denature

Question 6

Capillary electropheroesis

Answer 6

• Each sample gets a DNA size standards
• denature DNA
• detects labeled ss products

Question 7

Quality control fragments (4)

Answer 7

1. Benchmark fragment: 92 nucleotide long
   - ensures fragments are being detected properly
2. Quantity fragments:
   - ensures correct amount of sample DNA was added
3. D fragments:
   - ensureds denaturation was complete
4. X and Y fragments:
   - can identify sample mix-ups

Question 8

Other Quality control (3)

Answer 8

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