Methylation Specific Multiplex Ligation Probe Amplification

that is used to detect copy number variation (Schouten et al. 2002) (Figure 2.5). The technology involves the use of oligonucleotide probes of variable, but unique, lengths complimentary to various DNA targets. Other than the target specific sequence, the MLPA probes will also have identical 5‟ or 3‟ end sequences that are then amplified

by a single fluorescent primer pair. The products are analysed by capillary electrophoresis and different product sizes will reflect the different targets. In methylation specific MLPA (MS-MLPA), the addition of a digestion step using methylation sensitive endonucleases result in the ability to semi-quantify the methylation level in DNA samples (Nygren et al. 2005) (Figure 2.5). MS-MLPA has several advantages over other methylation analysis technologies; it is not based on bisulphite conversion, it requires a small amount of DNA, and it allows the multiplexing of several markers in a single reaction (Jeuken et al. 2007). The MS- MLPA kit ME042-A1 was used in this project to determine the methylation status of 8 markers (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) in paired normal and tumour DNA samples. All the steps were carried

according to the manufacturer‟s instructions (MRC-Holland, Amsterdam). It is noteworthy here that copy number analysis using the MS-MLPA kit was not performed, since the reference probes targeted unstable genomic areas in CRC.

MS-MLPA probes contain 2 oligonucleotides; one short synthetic and one long M13-derived. Up to 50 MS-MLPA probes can be used in a single reaction. Both oligonucleotides contain universal primer sites and target specific sequences. Each M13 derived oligonucleotide also contains a “stuffer” sequence with a probe specific length. Moreover, MS-MLPA probes also contain an HhaI recognition site (only methylation specific probes). When attached to a methylated DNA target, methylation sensitive HhaI enzyme will fail to digest the probe, a fluorescent signal will be produced, and peaks will be observed through the genemapper analysis. When attached to unmethylated DNA target, HhaI will digest the probes and no signal will be detected. *Adapted from (Nygren et al. 2005).

Universal forward primer Universal reverse primer

Variable size stuffer

Targets specific sequence

M

M

Methylated target Unmethylated target

Target DNA denaturation and multiplex probe hybridisation

M M

Simultaneous ligation & digestion with HhaI

PCR amplification with universal primers

Capillary electrophoresis & methylation analysis

Highly methylated DNA Non-methylated Figure 2.5 MS-MLPA technology

2.2.12.1 DNA denaturation and MS-MLPA probe hybridisation DNA samples (50 ng) were diluted in 5 μl of molecular biology grade 1xTE buffer (Promega) in 0.2 ml PCR tubes. The diluted DNA samples were then denatured by incubation in the GeneAmp® PCR System 9200 thermocycler (Applied Biosystems) at 98oC for 10 min followed by cooling down to 25oC. Following that, 3.0 μl of SALSA probe/MLPA buffer (1:1) mix were added to the samples, which were then mixed carefully by pipetting up and down and incubated at 95oC for 1 min followed by 60oC for 16 hrs.

2.2.12.2 Ligation and digestion

Before the end of the 16 hr incubation, the following mixes (per sample) were prepared and kept on ice; Ligase buffer A mix: 3 μl of ligase buffer A with 10 μl of nuclease free water, ligase-65 mix: 1.5 μl of ligase-65 buffer B, 0.25 μl of ligase-65 enzyme and 8.25 μl of nuclease free water, ligase-digestion mix: 1.5 μl of ligase-65 buffer B, 0.25 μl of ligase-65 enzyme, 0.5 μl HhaI enzyme (10 U/ μl) and 8.25 μl of

nuclease free water. For each sample, a new 0.2 ml PCR tube was labelled and prefixed with D (referring to digestion mix). After the end of the 16 hr hybridisation, 13 μl of ligase buffer A mix were added to each sample tube and mixed by pipetting. After that, 10 μl of the sample/ligase buffer A mix were transferred to the D prefixed

labelled PCR tubes. Both tubes were then incubated at 49oC for at least 1 min (in the thermocycler). While at 49oC, 10 μl ligase-65 mix were added to the first tube of each sample (ligation reaction), then 10 μl of the ligase-digestion mix were added to the D

prefixed labelled tubes of each sample (ligation digestion reaction). Both tubes were then incubated at 49oC for 30 min followed by 5 min at 98oC.

2.2.12.3 Amplification and capillary electrophoresis

For each sample, 2 PCR tubes (0.2 ml) were labelled (with and without the D- prefix). The following mixes were prepared and kept on ice; SALSA PCR buffer mix: 2 μl of SALSA PCR buffer with 13 μl nuclease free water, polymerase mix: 1 μl SALSA PCR

primers, 1 μl SALSA enzyme dilution buffer, 0.25 μl SALSA polymerase and 2.75 μl nuclease free water. In the new labelled 0.2 ml PCR tubes, 5 μl of MLPA ligation or 5 μl of ligation digestion reaction were mixed with 15 μl of SALSA PCR buffer mix. The

tubes were placed on ice and 5 μl polymerase mix was added to each tube. The tubes were then placed in a preheated thermocycler (72oC) and amplified using the following PCR conditions; 35 cycles of denaturation at 95oC for 30 sec, annealing at 60oC for 30 sec and extension at 72oC for 60 sec followed by 20 min incubation at 72oC. The PCR products were then electrophoresed on an ABI PRISMTM 3730 DNA Analyser with appropriate size standards by the Core Sequencing Facility.

2.2.12.4 MS-MLPA results analysis

The initial analysis of the MS-MLPA products was performed using genemapper software version 4.0 (Applied Biosystems). The raw data was then exported as a text file and the rest of the analysis described in Sections 2.2.12.4.1 - 2.2.12.4.3 was performed in Microsoft Excel spreadsheets templates and using an in-house algorithm designed by Dan Connely (Bioinformatician in our research group).

2.2.12.4.1 Quality control

Two types of quality control (QC) fragments are included in the MS-MLPA kit, the “Q” and the “D” fragments. The Q-fragments (64, 70, 76 and 82bp) are generated by the

Q-probes in a non-ligation dependent manner. The Q-peaks areas should be smaller

than half the area of D-peaks (below), if they were not, then the sample ligation failed or the DNA concentration was very low. The D-fragments (88, 92 and 96bp) are generated by the D-probes in a ligation dependent manner. The D-peaks areas should be comparable to other MLPA reference probes areas (≥40%) which mean that the ligation was successful, there was a sufficient amount of DNA and that they were properly denatured. If the 88 and the 96 D-fragments averaged areas were <40% of the 92 D-fragment area then denaturation was not enough. If the area of the 92 D-fragment was <40% of the 88 and 96 D-fragments averaged areas, it indicates

that hybridisation was incomplete. This can result from short hybridisation time, low hybridisation temperature, insufficient amounts of MLPA probe mix and/or MLPA buffer, the use of >5ul of DNA, or the thermal cycler lid temperature was less than 100oC. The QC tests are summarised in Table 2.6. Finally, a digestion control probe is included in the probe mix (Section 2.2.12.1) to test the efficiency of the digestion step. This probe should not give a signal upon digestion.

Table 2.6 MS-MLPA “Q” and “D” fragments QC tests

Test Description Pass* Fail*

A DNA Concentration/Ligation Q-peaks against D-peaks ≤50% >50%

B Denaturation 88D and 96D against 92D ≥40% <40%

C Hybridisation 92D against 88D and 96D ≥40% <40%

D DNA Quantity/Ligation/Denaturation D-peaks against reference peaks ≥40% <40% * The % reflect the ratio between the peaks areas

2.2.12.4.2 Intra-sample data normalisation

For the analysis of the MS-MLPA results, relative peak areas are used instead of absolute peak areas (generated by the ABI PRISMTM 3730 DNA Analyser). In order to calculate the relative peak areas, the data was normalised in an intra-sample fashion. This involved dividing the peak area generated from each marker probe by the peak area from each of the 10 reference probes in the same sample, thus creating 10 ratios for each of the marker probes. The median of the 10 marker probes ratios is considered the normalisation constant of the probe and is used for the methylation and copy number analyses. This normalisation was performed for the undigested as well as the digested samples.

2.2.12.4.3 Methylation analysis

The methylation status of the marker probe regions is calculated by dividing the normalisation constant of the probes from the digested sample by the normalisation constant of the probes from the corresponding undigested sample and multiplying the result by 100 to obtain the methylation percentage. In order to identify aberrant methylation, the tumour DNA methylation percentages were compared to their

matching normal DNA controls. A marker is considered to be methylated in the tumour DNA if the averaged methylation levels of all of its probes were greater by ≥10% than the value for the normal DNA. However, at least 2 probes should indicate

such an increase in methylation levels. This level of increase was previously shown to correlate with a marked decrease in mRNA expression (Cheng et al. 2008). Finally, a sample was classified as CIMP negative (CIMP-N) if none of the markers were methylated, CIMP low (CIMP-L) if 1-4 of the markers were methylated and CIMP high (CIMP-H) if 5 or more were methylated (Ogino et al. 2007).

2.2.12.4.4 Sex determination and BRAF V600E mutation

In addition to copy number and methylation analysis, the MS-MLPA kit ME042-A1 provides X and Y chromosome specific probes which can be used to determine the gender of the samples. Moreover, a mutation-specific probe that binds only in the presence of the BRAF mutation V600E is available.