MLPA

Question 1

Who created the MLPA technology?

Answer 1

MRC Holland

Question 2

What is MLPA?

Answer 2

Multiplex ligation-dependent Probe Amplification

Question 3

What is MLPA used for?

Answer 3

Identify copy number changes at an exon level

Question 4

Outline the 6 steps of MLPA:

Answer 4

Denaturation, Hybridization, Ligation, Amplification, Fragment Separation and data analysis

Question 5

MLPA is based on the amplification of what?

Answer 5

Probes

Question 6

Only [ ] probes are amplified

Answer 6

Ligated

Question 7

How is fragment separation visualized?

Answer 7

Capillary electrophoresis

Question 8

In what format is the fragment separation data visualized?

Answer 8

Electropherogram

Question 9

What happens during denaturation?

Answer 9

Double-stranded DNA is heated to become single-stranded using a thermocycler.

Question 10

What is added to the sample during the hybridization step?

Answer 10

Hybe master-mix

Question 11

What does the hybe master-mix consist of?

Answer 11

Salsa MLPA probe-mix and Salsa MLPA buffer

Question 12

What is the purpose of the Salsa MLPA buffer?

Answer 12

To create optimal conditions for hybridization

Question 13

How many different probes can be included in the Salsa MLPA probe-mix?

Answer 13

60 different MLPA probes each targeting a specific DNA sequence

Question 14

Each MLPA probe consists of [ ] pieces of single-stranded DNA, a left and a right probe oligonucleotide..

Answer 14

2

Question 15

Where do the left and right probe oligonucleotides bind in relation to the target sequence on the sample DNA?

Answer 15

Immediately adjacent

Question 16

How big is a typical target site?

Answer 16

Between 60 and 80 nucelotides in length.

Question 17

What is on the right oligonucleotide of the MLPA probe?

Answer 17

Stuffer sequence

Question 18

What is the purpose of the stuffer sequence?

Answer 18

To help determine the total length of the probe, ensure the probe is identifiable by length

Question 19

What is a stuffer sequence?

Answer 19

A sequence motif of unique length. Each probe has

Question 20

What enzyme is contained in the ligation master-mix?

Answer 20

Ligase 65

Question 21

What is the purpose of the Ligase 65 enzyme?

Answer 21

Catalyze the creation of a covalent bond between the right and left oligo.

Question 22

How is the ligation process terminated?

Answer 22

Heating the mixture inactivates the Ligase 65 enzyme.

Question 23

Describe the specificity of the Ligase 65 enzyme…

Answer 23

Ligase 65 is highly specific therefore if there is a mis-match between the target DNA sequence and the oligo sequence, ligation will not take place.

Question 24

Describe 2 ways in which the specificity of the Ligase 65 is useful:

Answer 24

1. Distinguish between a gene and a very similar pseudogene.
2. To detect specific point mutations.

Question 25

What does the Polymerase master mix contain?

Answer 25

Polymerase and primer-mix

Question 26

What primers are contained in the primer-mix?

Answer 26

Forward and reverse

Question 27

In addition to forward and reverse primers, what else is contained in the primer-mix?

Answer 27

dNTPs

Question 28

Which primer is fluorescently labelled?

Answer 28

Forward

Question 29

What happens to MLPA probes after ligation?

Answer 29

Exponentially amplified using a single primer pair

Question 30

How many times is the PCR cycle repeated?

Answer 30

x35

Question 31

Outline the 3 main steps of PCR amplification

Answer 31

1. Denaturation
2. Primer annealing
3. Primer elongation