18.02.05 MLPA Flashcards
Give a brief summary of the principle of MLPA.
- DNA is hybridised to probe sets
.2. The target specific sequences are located directly adjacent to one another, therefore when the probes bind to the DNA they can be joined together using a ligase. - This generates a contiguous probe flanked by universal primers.
- All ligated probes can then be amplified in a single PCR reaction using the same universal PCR primers.
- It is therefore the probes and not the target sequences that are amplified.
- Unbound probes will not be amplified, because they only contain one primer sequence.
- The amount of ligated probe produced is proportional to the copy number of the target region. Following PCR amplification, comparing the relative peak heights to a control sample can indicate changes in copy number.
Describe a MLPA probe set.
Each probe set consists of 2 halves:
1st half consists of target specific sequence (20-30 nucleotides) (blue) flanked by a universal primer (black).
2nd half consists of target specific sequence (25-43 nucleotides) (blue) flanked by a universal primer (black) BUT in-between is a random fragment of between 19-370 nucleotides, this is known as the stuffer sequence (green). The stuffer sequence is a different length for each probe pair and allows for the generation of different sized products for electrophoretic resolution
Describe the MLPA technique.
- Denaturation of the genomic DNA
- Hybridisation of the probes to the sample.
- Ligation of the probes
- PCR amplification of the ligated probes using universal primers
- Separation of the amplified products by capillary electrophoresis
- Analysis and quantification
What controls should be used in a MLPA reaction?
For each reaction reference/control samples should be included to normalise against, it is also recommended to run negative and positive controls and a “blank” to aid in interpretation and troubleshooting
Control fragments for DNA amount (Q fragments), denaturation, and X and Y markers are included.
What are the typical analysis parameters for autosomal MLPA targets?
After capillary electrophoresis the quality of the data should be assessed and then the ratios for each probe calculated using data analysis software. Abnormal results should be confirmed by repeating or by another method.
Expected ratios when comparing a patient and normal control DNA are:
0.5 (range 0.3-0.7) Heterozygous deletion
1 (range 0.7-1.3) Normal
1.5 (range 1.3-1.7) Heterozygous duplication
What is the application of MS-MLPA?
Allows copy number detection and methylation profiling. Can be used for imprinting disorders e.g. PWAS.
Describe the MS-MLPA process.
- MS-MLPA probes for methylation detection resemble other MLPA probes, except that their target sequence contains a restriction site for a methylation sensitive enzyme (HhaI)
- As with standard MLPA the DNA is denatured, MS-MLPA probes added and then hybridised for 16hrs.
- The probe-DNA mix is then split into two tubes – one for copy number detection and one for methylation profiling.
- One tube is processed as standard MLPA to provide information on the copy number.
- The other tube is incubated with a methylation specific enzyme whilst the probes are being ligated.
- Unmethylated DNA will be digested by the enzyme and therefore not amplified at the PCR stage and will not generate a signal. Methylated DNA will not be digested and the ligated probe will generate a signal.
Describe the analysis of MS-MLPA.
Analysis of MS-MLPA consists of two parts
1. Determining copy number by comparing different undigested samples
2. Determining methylation patterns by comparing each undigested sample with the digested sample, this gives a semi-quantified amount of methylation.
What is the application of RT-MLPA?
Reverse transcriptase MLPA is used for mRNA expression profiling.
Changes in gene expression are important in cell development and differentiation. This is quicker and more efficient than other methods such as northern blotting or real time PCR.
Kits available for apoptosis and inflammation genes.
What are the differences between MLPA and RT-MLPA?
The process is very similar to standard MLPA, however, ligase cannot ligate probes bound to RNA, and therefore reverse transcriptase is required to create cDNA from the RNA.
The MLPA reaction then continues as usual using cDNA instead of genomic DNA
What are the different methods of CNV detection using NGS?
- Read pair (RP)
- Split read (SR)
- Read depth (RD)
- Assembly
What are the pros and cons of using split reads for CNV detection?
SR can detect the exact breakpoints of SVs. However, it is limited to the length of the reads and NGS data shorter than 1 kb affect the accuracy and precision. In addition, SR is currently reliable only in the unique regions of the genome
Poor performance in regions enriched with duplications since they rely on confident and independent mapping of each end
What are the pros and cons of using read pairs for CNV detection?
RP is able to identify almost all types of SVs, but it is unable to detect the exact breakpoints with loose fragment size distributions. The accuracy of RP methods is largely dependent on the insert size. While small events can be missed with large-insert libraries, insertions larger than the library insert size might be ignored
Poor performance in regions enriched with duplications since they rely on confident and independent mapping of each end
In the RP approach, resolving ambiguous mappings in repetitive regions is challenging and accurate prediction of SV breakpoints depends on fragment size distributions, which can result in costly and complicated library construction
What are the pros and cons of using assembly for CNV detection?
AS generates a long sequence from the short reads, called contig/scaffold, that match the reference genome. However, it has been shown that AS has a poor performance against duplications or repeats
What are the pros and cons of using read depth for CNV detection?
RD is more reliable for regions with deletions and duplications and can also count the number of CNVs. However, similar to RP, it is difficult to identify the exact breakpoints in RD. Compared with RP, it is anticipated that RD events are enriched in segmental duplications
Although RD is the only method to accurately predict absolute copy numbers, the breakpoint resolution is often poor
