An outline of the MLPA reaction is shown in Figure . DNA (20–500 ng) is denatured and fragmented by a 5 min heat treatment at 98°C. MLPA probes are added and allowed to hybridise for 16 h at 60°C in a thermocycler with a heated lid. Dilution buffer including the ligase enzyme is added and ligation is allowed to proceed for 15 min at 54°C. After heat inactivation of the ligase and addition of PCR primers, dNTPs and polymerase, PCR amplification of the ligation products is started. One PCR primer is fluorescently or isotopically labelled. Amplification products are detected and quantified by capillary electrophoresis (see Figs –) or traditional gel electrophoresis (Fig. ).
Figure 4 Detection of trisomies by MLPA. Samples containing 100 ng DNA were analysed by MLPA using probe mix P001. Male and female control DNA was obtained from Promega. Blood-derived DNA from a triple X and a female triple 21 individual were provided by the (more ...)
Figure 8 MLPA analysis of SNPs/mutations resulting in amplification products for both alleles with different lengths. (Top) Schematic drawing. Target sequences A and B differ in 1 nt. The short synthetic probe oligonucleotides S1 and S2 differ also in (more ...)
Figure 3 Sensitivity of MLPA analysis to mismatches in the short probe oligonucleotide. MLPA reactions were performed on 100 ng samples of human DNA. Amplification products were separated on a denaturing acrylamide gel (LICOR). Length (bp) as well as gene HUGO (more ...)
Preparation and design of MLPA probes
Ligation-dependent PCR has been described previously (13
). Several modifications were made to make the technique simple to perform, more reproducible and sensitive, and suitable for true multiplex analysis.
Each MLPA probe consists of two oligonucleotides that can be ligated to each other when hybridised to a target sequence. All ligated probes have identical sequences at their 5′ and 3′ ends, permitting simultaneous amplification in a PCR containing only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. One of the two oligonucleotides of each MLPA probe is chemically synthesised and has a common sequence used for PCR amplification at the 5′ end and a target-specific sequence at the 3′ end. Each second probe oligonucleotide contains a sequence of 25–43 nt at the 5′ end that is able to hybridise to the target sequence immediately adjacent to the first probe oligonucleotide, a common sequence used for PCR amplification at the 3′ end, and a stuffer sequence of 19–370 nt in between. Chemically synthesised oligonucleotides of this size (80–440 nt) are not commercially available in the quality needed for MLPA. We therefore use single stranded DNA from M13 clones containing small target-specific sequences for the preparation of these oligonucleotides. The M13 DNA is made partially double stranded by the annealing of complementary oligonucleotides and is digested by two restriction endonucleases (Fig. ). One of these enzymes cuts the DNA outside its recognition sequence, resulting in a 5′ phosphorylated end that is perfectly complementary to the target sequence. Most probe mixes made contain 35–42 probes with length differences between consecutive amplification products of 6 or 9 bp. All probes used in a specific MLPA probe mix are made in a different M13-derived vector and have different stuffer and hybridising sequences. Amplification products of different probes have common sequences only at their ends in order to prevent heteroduplex formation during later stages of the amplification reaction when competition between duplex formation and PCR primer annealing takes place. We prepared a set of 118 different M13-derived MLPA vectors, each containing a stuffer sequence of different length and sequence. Target sequence-specific synthetic oligonucleotides can easily be inserted in these vectors, allowing flexibility to create all required fragment lengths. Probes consisting of two synthetic oligonucleotides and resulting in amplification products of 94–124 bp were also successfully used.
The non-hybridising stuffer sequences of our M13-derived probe oligonucleotides provide the advantage that the amplification characteristics of a large part of each amplicon are known. Short hybridising sequences also have an advantage in mutation detection and SNP analysis as sequences close to each other can be analysed without competition between probes with overlapping target sequences.
All M13-derived MLPA probe oligonucleotides contain the complement sequence of one of the two PCR primers at their 3′ end, and will thus be linearly amplified during the PCR. This PCR primer is unlabelled in order to prevent background signals. To prevent most of this PCR primer being consumed by linear amplification, low amounts of probe oligonucleotides have to be used. If 10 fmol of each of the 40 probes were to be linearly amplified, this would consume all 10 pmol unlabelled primer in only 25 PCR cycles. Despite the use of low amounts of probes, hybridisation of probe oligonucleotides to their target sequences has to be complete in order to obtain reproducible results. We use a 16 h hybridisation period in an 8 µl reaction volume containing 1–4 fmol of most probe oligonucleotides. Hybridisation kinetics differed slightly for each oligonucleotide. Some probes required the presence of up to 8 fmol. Once hybridisation of all probes is complete, prolonged incubation did not influence relative probe signal strength.
Commercially available ligases, as well as several ligases cloned at MRC-Holland, were tested for use in MLPA reactions. The NAD requiring Ligase-65 enzyme chosen is active at 50–65°C, but can easily be heat inactivated before the start of the amplification reaction. Ligase-65 is very sensitive to probe-target mismatches next to the ligation site (Fig. ). Within the range of 20–500 ng human sample DNA, MLPA results were not influenced by the amount of DNA used. Some non-specific amplification products appeared when samples containing <20 ng human chromosomal DNA were analysed. Most experiments were performed using 50–100 ng human sample DNA.
Probe signal strengths
The hybridising parts of our probes were designed to detect human sequences that are present in a single copy/haploid genome. However, the relative signal strength of different probes (relative peak area of each probe amplification product) is not equal. Apart from the copy number of the probe target sequence, the major factors influencing the relative signal strength of MLPA probes proved to be the amount of polymerase used in the PCR and the nature of the first nucleotide following the labelled PCR primer. The MgCl2 concentration in the PCR did not have substantial effects. The KCl concentration, however, influenced the relative peak sizes of some probe amplification products.
The polymerase activity during the PCR influenced the relative signal strength of a minority of the probes. Relative peak areas of 5–10% of the probes made decreased >25% when 2.5-fold lower amounts of polymerase were used, whilst the relative peak areas of 2% of the probes strongly decreased when 2–4 times higher amounts of polymerase were used. These probes were replaced.
The influence of the nature of the first nucleotide following the PCR primer sequence on the relative peak size was unexpected. In our short synthetic probe oligonucleotides the PCR primer sequence is immediately followed by the variable target-specific sequence. Probes in which the PCR primer sequence was followed by an adenine had a >2-fold lower average signal strength. In all 25 cases examined, replacement of this A nucleotide resulted in increased signal strength. Signal strength increased in the order A < T < G < C. Average signal strength of probes in which the unlabelled primer was first elongated with an adenine residue was also significantly lower as compared with other probes. This finding may be of use for the design of primers for traditional multiplex PCR. The vast majority of recently made MLPA probes, designed to detect human single copy DNA sequences on chromosomes 1–22, have signal strengths between 50 and 150% of the average signal strength. This indicates a maximum difference in amplification efficiency during each PCR cycle of <2% from average for each probe amplification product.
As shown in Figure , omitting one of the short probe oligonucleotides, or replacing it by an identical oligonucleotide having a mismatch at the 3′ end, prevented the appearance of probe amplification products. Sensitivity of probe signals to mismatches close to the ligation site depended on the amount of ligase used and the duration of the ligation reaction (not shown). Under our experimental conditions a mismatch at the 3′ end of the short probe oligonucleotide completely prevented the appearance of probe amplification products. Mismatches at 4–6 nt from the ligation site had no or only small effects (Fig. ). This sensitivity to a mismatch at the ligation site was, in some cases, used to distinguish target sequences from pseudogenes or related genes.
Detection of trisomies
In order to test the linearity of probe signal with small changes in target sequence copy number, we prepared a mix of 40 probes, including four probes each for chromosomes X and Y and eight probes each for chromosomes 13, 18 and 21 sequences. Part of the capillary electrophoresis patterns obtained on four DNA samples are shown in Figure . For relative quantification purposes we divided each peak area by the sum of all probe peak areas of that sample. The ratio of each individual probe relative area was then normalised to that obtained on a control sample. In Table , this ratio is shown for each probe using female control, triple X, triple 13, triple 18 and Down’s syndrome (trisomy 21) DNA. Results show that the relative probe signals obtained for each probe reflected the relative amount of the probe target sequences in the sample. The excellent reproducibility of relative signals obtained enabled the detection of a single extra copy of a probe target sequence per diploid genome. Highest deviations of the expected values were obtained for the triple 13 DNA sample that was derived from a cell line. An increase in relative peak area of the chromosome 8-specific MYC probe (1.30), and a lower than expected relative peak area of the chromosome 13-specific RB1 (1.30) and DLEU 1 probes (1.25) might be due to chromosomal aberrations in some cells of our cell line. The DLEU1 gene and the tumour suppressor RB1 are located close (<1 Mb) to each other. A probe specific for a different sequence of the MYC gene (relative signal 1.35) also indicated an increase in copy number of this oncogene in our triple 13 cell line.
Detection of exon deletions in the human BRCA1, MLH1 and MSH2 genes
The human BRCA1 gene is involved in hereditary disposition for breast cancer. In The Netherlands >30% of hereditary disposition for BRCA1-related breast cancer is due to deletions of one or more of the 24 BRCA1 exons (2
). Deletion of one or more exons can be proved by Southern blotting provided that the deletion does not extend beyond the probe boundaries. Known deletions can be tested by PCR. We prepared MLPA probes for each BRCA1 exon. Samples known from specific PCRs (2
) to be heterozygote for a deletion of either exon 13 or exon 22 were easily identified by MLPA as they resulted in an ~2-fold reduction of relative probe signal for these probes (Fig. ). DNA samples from 850 individuals suspected of hereditary disposition for breast cancer have been tested by MLPA. Several new as well as several previously described aberrations of the human BRCA1 gene were detected (F.B.L.Hogervorst, P.M.Nederlof, J.J.P.Gille, C.J.McElgunn, M.Grippeling, R.Pruntel, R.Regnerus, T.van Welsem, F.H.Menko, I.Kluijt, C.Dommering, S.Verhoef, J.Schouten, L.J.van ’t Veer and G.Pals, manuscript in preparation).
Figure 5 Detection of BRCA1 exon deletions by MLPA. Samples containing ~100 ng DNA were analysed by MLPA using probe mix P002. Female control DNA was obtained from Promega. Blood-derived DNA from individuals known to contain an exon 13 or an exon 22 deletion (more ...)
Genomic deletions of parts of the human MLH1 and MSH2 genes are a frequent cause of hereditary non-polyposis colon cancer (HNPCC) (3
). An MLPA probe mix was prepared containing probes for each of the 19 MLH1 exons and each of the 16 MSH2 exons, as well as seven probes for genes on other chromosomes. Using this probe mix, all six different known exon deletions tested could easily be identified by MLPA. Screening of a large number of DNA samples from HNPCC patients is in progress. Results obtained on a sample known to contain in one chromosome a deletion of exons 1–6 of the MSH2 gene are shown in Figure .
Figure 6 Detection of MSH2 exon deletions by MLPA. Samples containing ~100 ng DNA were analysed by MLPA using probe mix P003. Female control DNA was obtained from Promega. Blood-derived DNA from an individual known to contain a deletion of exons 1–6 (more ...)
Detection of gains and losses of chromosomal regions
For analysis of tumour DNA, three MLPA probe mixes have been made, each containing 41 different probes specific for human single copy DNA sequences. Most target sequences were chosen in chromosomal regions often deleted or amplified in various types of tumours. These probe mixes were used to analyse DNA from diffuse large B-cell lymphomas (DLBCL) as well as DNA from the SkBr3 cell line. Results obtained with one of the probe mixes on a DLBCL DNA sample and the SkBr3 DNA are shown in Figure .
Figure 7 Detection of gains and losses of human chromosomal sequences by MLPA. Samples containing ~100 ng DNA were analysed by MLPA using probe mix P006. Male control DNA was obtained from Promega. DNA isolated from a frozen DLBCL lymphoma was provided (more ...)
SkBr3 is known to contain amplified ERBB2 (HER2/neu) and MYC loci (16
). Relative peak areas for two different MYC-specific MLPA probes were increased 5.3 and 6.1 times compared with control human DNA. Relative peak areas of three different ERBB2 probes were increased 5.3, 6.5 and 6.0 times, respectively. This compares well with the 6.6 times amplification of ERBB2 in SkBr3 as measured by FISH (4
). Exact amplification levels are difficult to determine as the number of chromosomal aberrations is large and the relative copy numbers are calculated by comparison with the total peak area of a sample. The SkBr3 cell line is known to be substantially tetraploid and to contain several abnormal chromosomes (17
). Apart from many other aberrations, our SkBr3 DNA sample completely lacked the target sequence for a CDH1 probe on chromosome 16q22. Major parts of chromosomes 7 and 20 appeared to have a 1.5–2-fold increased relative copy number whereas BRCA1-specific probes had a 2-fold lower relative peak area.
The DNA from the (male) DLBCL lymphoma showed a 2.4–3.0-fold increase in relative peak area for each of three different BCL2-specific probes. Probes for DCC and PMAIP1, which are located on chromosome 18 within 11 Mb of BCL2, were also increased 2–3-fold whereas CDH2, which is located 37 Mb from the BCL2 locus, was not increased. Probes for CDKN2A and CDKN2B were both reduced by 82%, whereas two chromosome Y probes were reduced by 73 and 82%, respectively, indicating homozygous deletion of 9p21 and chromosome Y in the tumour and a high percentage of tumour cells in the sample. All seven probes surrounding the HLA region on chromosome 6p had a 25–56% reduction in relative peak area.
The same probe mixes were used to analyse 15 DNA samples extracted from formalin-treated paraffin-embedded primary breast cancer tissues. Reversal of formaldehyde cross-linking was performed by prolonged heating of the DNA (18
). Most of the results obtained by MLPA corresponded with results obtained on these samples by CGH (not shown).
SNP and mutation detection
MLPA probe signal is completely absent if the short probe oligonucleotide has a mismatch at the 3′ nucleotide when annealed to the target sequence (Fig. ). This sensitivity of the ligase for a mismatch next to the ligation site can be used to distinguish two sequences differing in only one or a few nucleotides, such as SNPs and various mutations. In order to obtain a signal from both alleles, which can be distinguished by the length of the probe amplification products, we used probes for the two alleles that have the M13-derived oligonucleotide in common, as outlined in Figure . Two different short synthetic probe oligonucleotides are used that differ at both the site of the mutation/SNP and also by 3 nt in length. These two oligonucleotides will compete with each other for binding to both target sequences as a single mismatch at the end of the probe oligonucleotide will usually not be sufficient to destabilise the hybrids. However, ligation of hybrids with mismatches next to the ligation site will occur with very low efficiency. In Figure , MLPA results are shown in which a DNA sample of a cystic fibrosis patient homozygote for the ΔF508 mutation is compared with samples from a heterozygote and a control DNA. The probe designed resulted in a 3 bp longer amplification product for the ΔF508 mutation as compared with the probe for the wild-type DNA sequence.