MS-MLPA

Question 1

MS-MLPA

Answer 1

• semi-quantitative method
• determines whether template DNA is methylated
• probes designed to hybridize where target DNA includes a CpG methylation restriction enzyme site (Hha1)

Question 2

DNMTs

Answer 2

-DNA methyltransferases
- catalyzes the transfer of a methyl group from S-adenyl methionine (SAM) to the 5th carbon of cytosine residue = forms 5-methylcytosine

Question 3

Transcription is ultimately regulated by the interaction of multiple __ mechanisms that cooperate to __ or __ gene expression

Answer 3

Transcription is ultimately regulated by the interaction of multiple EPIGENETIC mechanisms that cooperate to ACTIVATE or SILENCE gene expression

Question 4

Methylation is regulated by proteins such as __ and__ that are involved in the active addition or chemical modification of DNA methylation

Answer 4

Methylation is regulated by proteins such as Dnmt and Tet that are involved in the active addition or chemical modification of DNA methylation

Question 5

How do Dnmts suppress gene expression ?

Answer 5

Dnmt targets CpG sites and actively methylates DNA

Question 6

Hha1

Answer 6

• a restriction endocnuclease*
• recognizes GCGC sequence
• activity is blocked by methylation of first cytosine

*NOTE: cuts at the middle of DNA segments, not at ends

Question 7

Describe Denaturation in MS-MLPA

Answer 7

98 °C for 5 min = dsDNA template separates into ssDNA

Question 8

MS-MLPA Tube 2

Answer 8

• Ligation & Hha1 Digestion
• Unmethylated and methylated strands get ligated
• Unmethylated GCGC strand is cleaved by Hha1
• Methylated GCGC are protected from cleavage

Question 9

Products from Multiplex Ligation-dependent Probe Amplification are measured using __, along with __

Answer 9

Products from Multiplex Ligation-dependent Probe Amplification are measured using CAPILLARY ELECTROPHORESIS, along with DNA SIZE STANDARDS

Question 10

T or F: MS-MLPA can be used to identify Angelman and Prader-Willi Syndrome

Answer 10

TRUE; MS-MLPA can be used to identify Imprinting Disorders like Angelman and Prader-Willi Syndrome

Question 11

Where does Dnmt1 bind; what is its purpose ?

Answer 11

• binds at replication sites
• adds methyl groups to newly formed daughter strands to maintain methylation status as parent DNA strand

Question 12

MS-MLPA Probe Design

Answer 12

Left Probe:
- coding strand for primer binding site 2
- target specific sequence

Right Probe:
- Hha1 recognition site within target specific sequence
- stuffer* region
- primer binding site 1

Question 13

Describe the Two treatment Tubes in MS-MLPA

Answer 13

• after hybridization; probe specifically binds target DNA with Hha1 sequence

Tube 1:
- both methylated and un-methylated GCGC sites are ligated

Tube 2:
- both methylated and un-methylated GCGC sites are ligated
- digestion by Hha1: un-methylated GCGC strands are cut
- methylation protects GCGC strands

Question 14

Differentiate Amplification in MS-MLPA in Tube 1 vs Tube 2

Answer 14

• Tube 2 amplifies ligated, methylated target ONLY, NOT digested un-methylated

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