MLPA Flashcards
Who created the MLPA technology?
MRC Holland
What is MLPA?
Multiplex ligation-dependent Probe Amplification
What is MLPA used for?
Identify copy number changes at an exon level
Outline the 6 steps of MLPA:
Denaturation, Hybridization, Ligation, Amplification, Fragment Separation and data analysis
MLPA is based on the amplification of what?
Probes
Only [ ] probes are amplified
Ligated
How is fragment separation visualized?
Capillary electrophoresis
In what format is the fragment separation data visualized?
Electropherogram
What happens during denaturation?
Double-stranded DNA is heated to become single-stranded using a thermocycler.
What is added to the sample during the hybridization step?
Hybe master-mix
What does the hybe master-mix consist of?
Salsa MLPA probe-mix and Salsa MLPA buffer
What is the purpose of the Salsa MLPA buffer?
To create optimal conditions for hybridization
How many different probes can be included in the Salsa MLPA probe-mix?
60 different MLPA probes each targeting a specific DNA sequence
Each MLPA probe consists of [ ] pieces of single-stranded DNA, a left and a right probe oligonucleotide..
2
Where do the left and right probe oligonucleotides bind in relation to the target sequence on the sample DNA?
Immediately adjacent
How big is a typical target site?
Between 60 and 80 nucelotides in length.
What is on the right oligonucleotide of the MLPA probe?
Stuffer sequence
What is the purpose of the stuffer sequence?
To help determine the total length of the probe, ensure the probe is identifiable by length
What is a stuffer sequence?
A sequence motif of unique length. Each probe has
What enzyme is contained in the ligation master-mix?
Ligase 65
What is the purpose of the Ligase 65 enzyme?
Catalyze the creation of a covalent bond between the right and left oligo.
How is the ligation process terminated?
Heating the mixture inactivates the Ligase 65 enzyme.
Describe the specificity of the Ligase 65 enzyme…
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.
Describe 2 ways in which the specificity of the Ligase 65 is useful:
- Distinguish between a gene and a very similar pseudogene.
- To detect specific point mutations.
What does the Polymerase master mix contain?
Polymerase and primer-mix
What primers are contained in the primer-mix?
Forward and reverse
In addition to forward and reverse primers, what else is contained in the primer-mix?
dNTPs
Which primer is fluorescently labelled?
Forward
What happens to MLPA probes after ligation?
Exponentially amplified using a single primer pair
How many times is the PCR cycle repeated?
x35
Outline the 3 main steps of PCR amplification
- Denaturation
- Primer annealing
- Primer elongation
