Multiplex Ligation-dependent Probe Amplification (MLPA)

Contents:
 * The manuscript is based on the paper by Harteveld et al., J Med Genet (2005) ; 42:922–931.


 * The following protocol is for the diagnosis of &alpha;-thalassaemia deletions by MLPA as done in the Oxford laboratory.

Principle
Thalassaemias are inherited hemoglobin disorders characterized by a quantitative reduction of the &alpha;- or &beta;-globin chains [1,2,3]. Genomic deletions involving the &alpha;-globin gene cluster on chromosome 16p13.3 are the most common molecular cause of &alpha;-thalassemia (approx. 80-90% of cases). Rearrangements in the &beta;-globin gene cluster on 11p15.4 account for approx. 10% of all &beta;-thalassemia mutations and Hereditary Persistence of Fetal Hemoglobin (HPFH) syndromes. Besides the most common ones a large variety of less frequently occurring thalassemia deletions have been found in different populations. At least 60 different deletions involving the &beta;- and more than 50 involving the &alpha;-globin gene cluster have been described to date [4,5] (http://globin.cse.psu.edu/hbvar/menu.html).

The molecular tests commonly used to identify these deletions are gap-PCR, Southern blot analysis and Fluorescent In Situ Hybridization (FISH) analysis [6,7,8,9,10]. The gap-PCR can only be applied to known deletions. Southern blot is time consuming, technically demanding and the success is very much dependent upon the hybridization probes available. FISH involves laborious cell culturing to generate metaphase chromosome spreads and has a low resolution (>20kb).

Recently a simple technique suitable for rapid quantitative analysis has been described, called multiplex ligation-dependent probe amplification (MLPA) [11]. This technique is based on the ligation and PCR amplification of two adjacently hybridising oligonucleotides. Each oligonucleotide pair is designed to give a product of a unique length, and by using common ends all probes can be amplified with one primer pair. Using a fluorescent label allows probe separation on a capillary sequencing system. This method has been applied successfully in a number of genes in which deletions and duplications are common [12,13,14]. In the original description the probes were generated by cloning into specially developed M13 vectors. Recently we have simplified this method by using chemically synthesized oligonucleotides. Discrimination of probes based on chemically synthesized oligo’s (~40-60 nt) was doubled using two universal primer sets each labeled with a different fluorophore, allowing up to 40 probes to be used in a single reaction [15].

To simplify the detection of &alpha;- and &beta;-thalassemia deletions and increase the resolutions, we designed two probe sets for each cluster. For the &alpha;-cluster two probe sets of in total 35 probe pairs were designed with an average distance of ~20 kb, covering a genomic region of ~700 kb. For the &beta;-cluster a total of 3 probe sets consisting of 50 probe pairs were made covering a region of ~500 kb and an average distance of ~10 kb. Control DNA was used of known &alpha;- and &beta;-thalassemia deletion carriers for which the deletion was characterized by an independent method. Two groups of patient samples suspected of having a (large) deletion in either the &alpha;- or &beta;-globin gene cluster, based on hematological findings, were analyzed in this assay.

Patients
Patients suspected for hemoglobinopathies were sent to our laboratory for hematological, biochemical and DNA analysis [16]. Based on this analysis they were diagnosed as &alpha;- and/or &beta;-thalassemia carriers. The patients suspected for &alpha;-thalassemia in which no abnormalities were found by gap-PCR for the 7 most common -thalassemia deletions and non-deletion types of &alpha;-thalassemia were excluded by direct sequencing of the &alpha;-genes were selected for MLPA. Some showed either an unbalanced &alpha;/&beta; chain synthesis ratio (< 0.8) and/or Inclusion Bodies [17] indicative for a deletion of both &alpha;-genes on the same allele [18]. In addition, few patients presented with HbH disease, but analysis thus far only revealed one mutation, suggesting a deleted allele in trans. Some showed the presence of possible junction fragments by Southern blot, in which the deletion could not be characterized due to lack of probes in the region flanking the potential deletion. In total 38 possible &alpha;-thalassemia carriers were selected to be screened for rearrangements in 16p13.3. These samples were collected during a period of approximately 5 years.

Patients were selected presenting with a microcytic hypochromic anemia in the presence of elevated HbA2 levels, for which standard DNA analysis revealed no abnormalities in the &beta;-globin gene sequence or the 5’and 3’UTR. These samples include patients showing a high HbF expression, indicative for Hereditary Persistence of Fetal Hemoglobin, (&delta;&beta;)o- or G&gamma;(A&gamma;&delta;&beta;)o-thalassemia and patients showing normal HbA2 and HbF levels with &alpha;/&beta; chain synthesis ratios higher than 1.5, indicative for deletions involving the complete cluster and/or the regulatory elements. In total 51 samples were analyzed by MLPA.

As positive controls for MLPA of the &alpha;-globin gene cluster, we used seven deletions confirmed previously by gap-PCR (--SEA, -&alpha;3.7, -&alpha;4.2, -- Med I, -- FIL, -- THAI and -(&alpha;)20.5, indicated as black bars in Figure 1b). Two other deletions (33 kb --Dutch I and the -&alpha;7.9 ) were previously characterized by Southern blot analysis and direct sequencing of the amplified break point fragments [19,20]. For the MLPA of the &beta;-cluster the Dutch III (&epsilon;&gamma;G&gamma;A)o&delta;&beta;-thalassaemia of 112 kb [21], the 50 kb Belgian (&gamma;&delta;&beta;)o-thal [22], the 25-30 kb Chinese &beta;o-thal [23], the 12.6 kb Dutch I &beta;o-thal [24] and the Indian (-619 bp) &beta;o-thal deletions [25], all previously characterized by Southern blot analysis were used as positive controls (Figure 2).

Probe design
In total 35 probe pairs were designed to detect rearrangements on 16p13.3, covering approximately 700 kb from the telomere to the MSLN gene (Table 1, Figure 1). For each probe pair the common ends correspond to either the MLPA amplification primers (forward tag 5'-GGGTTCCCTAAGGGTTGGA-3'; reverse tag 5'-TCTAGATTGGATCTTGCTGGC-3') [11] or the MAPH primers (forward tag 5'-GGCCGCGGGAATTCGATT-3'; reverse tag 5'-CACTAGTGAATTCGCGGC-3') [26], which allows simultaneous amplification and detection of the separated fragments in different colors.

Similarly 34 probe pairs to be analyzed in two colors were designed to detect rearrangements in on 11p15.4 (Table 2, Figure 2). A third probe set, consisting of an additional 16 probe pairs, was designed for fine mapping some of the deletions found by MLPA (see Table 2 (continued)). In order to detect all 50 probe sets in the same fragment analysis sample run, a third common extension was used for the additional probe set, which allowed the use of a third color (M13 forward tag 5'-GGCGATTAAGTTGGGTAAC-3'; M13 reverse tag 5'-GTTCACACAGGAAACAGC-3').

Unique sequence was identified using the BLAT program (http://genome.ucsc.edu) [27], care was taken that no known sequence variants are present in the primer annealing site. Probes within each set were designed to produce PCR products differing by 2 bp in length to allow separation in the size range from 80 to 125 bp using capillary electrophoresis on the ABI 3700 (Applied Biosystems). Primers have been designed such that the Tm of the hybridizing regions of each probe was at least 65oC (RAW program, MRC-Holland, Amsterdam, Nederland), with a GC percentage between 35% and 60%.

The oligonucleotides were ordered from Illumina, Inc. (San Diego, CA), synthesized in a salt-free environment (50 nmol scale) and used without further purification. Of each probe pair the downstream primer was 5' phosphorylated to allow ligation. Separate probe mixes were prepared to allow the detection of deletions in either the &alpha;- or &gamma;-globin gene clusters, combining two sets of probes with MLPA and MAPH common ends at a final concentration of 4 fmol/&mu;l. The &alpha;- and &beta;-globin gene MLPA probe mixes are available on request (http://www.LGTC.nl).

MLPA Reaction
Reagents for MLPA and subsequent PCR amplification were purchased from MRC-Holland (Amsterdam, Nederland). All primers used for PCR amplification were purchased from Sigma-Genosys Ltd (UK). The MLPA reactions were performed as described by Schouten et al. [11] and White et al. [15]; in brief, approximately 200 ng of genomic DNA in a final volume of 5 &mu;l was heated for 5 minutes at 98oC. After cooling to room temperature, 1.5 &mu;l of the probe mix and 1.5 &mu;l SALSA hybridization buffer (MRC-Holland) were added to each sample, followed by heat denaturation (2 minutes at 95oC), hybridization (16 hrs at 60oC). Ligation was performed by adding 32 &mu;l of ligation mix at 54oC for 10 minutes and the reaction was stopped by incubating 5 minutes at 95oC. PCR amplification was carried out for 33 cycles in a final volume of 25 &mu;l, adding both the MAPH-F and –R and the MLPA-F and –R primer sets to a final concentration of 100 nM and 200 nM respectively, with MAPH-F being labeled with HEX and MLPA-F labeled with FAM. The third common primer set used for the beta-globin gene cluster is called M13-F and M13-R and the primers are labeled with ROX and were added to a final concentration of 100 nM. A size standard (0.05 &mu;l ROX 500, Applied Biosystems, www.appliedbiosystems.com) was added to each well and products were separated by capillary electrophoresis on the ABI 3700 (Applied Biosystems) (Figure 3).

Data analysis
For quantitative analysis, trace data from GeneScan (Applied Biosystems) were exported to Excel (Microsoft; www.microsoft.com) to calculate allelic loss in the patient samples tested [15]. In brief, two probes for unlinked loci were included per probe set as a reference in each sample. The height of each &alpha;- (or &beta;-) globin cluster specific probe peak was divided by the sum of the heights of the two reference probe peaks to give a ratio. The median ratio for each probe across all samples was calculated and this value was used to normalize each probe to 1.0, which corresponds to a copy number of two. The upper threshold for deletions was set at 0.75 and the lower threshold for duplications at 1.25. The normalizing factor for each sample was calculated as the mean value of the unaffected probes within a sample (defined as falling between 0.8 and 1.2) and dividing all values within that sample by this value.

All samples were tested at least twice. Detection of deletions is simplified by the fact that a series of flanking probes all generate a decreased signal. In cases of unlinked or single probe deletions the region covering the MLPA probes is amplified and sequenced to rule out the presence of rare sequence variants under the ligation site.

Design of the MLPA assay for &alpha;-thalassemia rearrangements
Fragment analysis in the size range of 80-125 bp allows the simultaneous amplification of approximately 20 probes differing 2 bp in length. To maximize the number of loci that can be analyzed in a single MLPA assay, we used a second primer set with common ends, to allow co-amplification of the two primer sets under the same PCR conditions. Probes were designed for each gene and pseudo-gene in the &alpha;-globin gene cluster, in the unique sequences L0 and L1, at the HS-40, the MPG gene and more proximal at conserved sequences respectively 20 and 9 kb from the MPG gene (Figure 1). More distally, two probes were designed flanking the 3'HVR, known to be involved in many rearrangements of the &alpha;-cluster, and 15 probes at approximate intervals of 13 to 50 kb with the most proximal probe localized in the MSLN gene, known to be deleted in the Alpha Thalassemia mental Retardation Syndrome (ATR-16) [28,29]. The 35 probe pairs shown in Table 1 can detect all of the deletions described to date.

Of the 35 probes tested in triplo on 14 healthy individuals, 2 gave a standard deviation of greater than 12% (Table 1, probes 17a and 21a). These probes were considered to be unreliable and excluded for further calculations. To investigate the efficacy of the assay DNA samples of nine carriers with known deletions were used as positive controls. All could be detected unequivocally and their extent could be confirmed (black bars in Figure 1b).

To demonstrate that also duplications are reliably detected, we tested a homozygote and heterozygote carrier for the common -&alpha;3.7 deletion, which results in the loss of the &alpha;2-specific 3'UTR and a heterozygote for the so called &alpha;-triplication, which is characterized by a duplication of the &alpha;2-specific 3'UTR. The results are summarized in Figure 3.

MLPA for &beta;-thal rearrangements and HPFH
Similar to the &alpha;-cluster, 34 probes were designed for loci in the &beta;-globin gene cluster and flanking regions. The region spans from the olfactory receptor gene OR52D1 to OR52A4 and covers an area of approximately 370 kb (Table 2, Figure 2). Most large deletions reported so far are located in this region and all should be detectable. In order to be able to detect small deletions taking away part of, or all of the &beta;-gene [30] a subset of closely spaced probes (Figure 2b) surrounding the &beta;-globin gene were selected. A third probe set was designed with different common ends (M13-F and –R) to allow amplification and detection with a third color. Loci were selected in between some widely spaced probes and towards the centromere. Standard deviations for these probe set was calculated on 19 healthy individuals, none showed standard deviations greater than 12%.

Positive controls (marked as black bars in Figure 2) were used to test the capacity of the MLPA assay to detect the deletions found by other methods in these patients. Probes covering deleted loci showed half the intensity of the surrounding probes, matching the positions and extensions of all of the 6 known deletions.

Patient samples for &alpha;-thalassemia
Our MLPA analysis revealed a large deletion involving the &alpha;-globin genes in 19 out of 38 patients. In the remaining 19 patients 11 different deletions were detected, affecting either the &alpha;-globin genes or the regulatory elements known to be involved in globin gene expression. Six showed no resemblance to previously described deletions and were considered to be new (-- GZ, --OH, (&alpha;&alpha;)L, (&alpha;&alpha;)ZW, -- AB, -- MK). One has been described (Dutch II &alpha;o-thal.) but the breakpoint position and deletion length could not be determined at the time [31], FISH analysis performed in the Radcliff Hospital in Oxford revealed an approximate deletion length of 300 kb (Higgs, personal communication). Four deletions show similarity with previously described deletions (Figure 1b, last 4 deletions). One 14 year old Dutch girl showed hematological parameters typical for an &alpha;o-thalassemia carrier (MCV 65 fl, MCH 19.5 pg, RBC 5.79x1012 and positive HbH Inclusion Bodies test). The &alpha;-genes were structurally intact and we only detected the deletion of a single probe 5a (Figure 1a (&alpha;&alpha;)ZW ). The location of this probe coincides with one of the the cis-acting elements that regulate &alpha;-gene expression, known as the HS-40.

Patient sample for &beta;-thalassemia
Analysis of the 51 samples suspected for &beta;-thalassemic rearrangements or HPFH using MLPA revealed 10 different deletions in 31 out of patient 51 samples. In the remaining 20 samples a deletion of the probe sets tested could be excluded. In three cases deletions were detected which do not match the ones described to date and are considered to be new. All three deletions, found in Dutch carriers, silence the expression of the complete globin gene locus and were named Dutch IV (&epsilon;G&gamma;A&gamma;)&delta;&beta;o -, Dutch V &epsilon;G&gamma;A&gamma;&delta;&beta;o- and Dutch VI (&epsilon;G&gamma;A&gamma;&delta;&beta;)o-thalassemia. One was matching the HPFH-2 deletion and was confirmed by breakpoint PCR [8]. One sample belonged to a patient described in 1996 by Abels et al. [32] as a carrier of the Dutch II (&epsilon;G&gamma;A&gamma;&delta;&beta;)o-thalassemia, however the deletion length was not determined at that time. Now the deletion length is estimated to be at least larger than 400 kb and the 5’breakpoint located between position 5408246 and 5387552 (UCSC Genome Browser, May 2004) (Figure 2a). Five deletions match the length and breakpoint locations of previously described deletions, two of which, the Croatian (&epsilon;&gamma;&delta;&beta;)0- (at least > 108 kb) and the Filipino &beta;o-thal (at least > 45 kb), were incompletely mapped. A more accurate length estimation was obtained by MLPA being between 128-143 kb and 109-122 kb respectively. The other three showed similarity to the Dutch I 12.6 kb &beta;o-thal deletion (in 7 independent patients of Dutch origin), the 13.4 kb Sicilian (&delta;&beta;)o-thal deletion, which are also frequently found in the Mediterranean basin [33,34] and the 32.6 kb Indian G&gamma;A&gamma;(&delta;&beta;)o-thal [35], found in four independent chromosomes from Surinam-Hindustani (Figure 2b.).

Discussion
We describe the application of MLPA for high resolution mapping of deletions causing &alpha;- and &beta;-thalassemia. Using synthetic oligonucleotides, 35 loci along a genomic region of 700 kb from the tip of the short arm of chromosome 16, containing the alpha-globin gene cluster, could be analyzed in two colors in a single reaction. An increase in the number of loci to be analyzed simultaneously was obtained by using a third pair of amplification primers, labeled with a 3rd fluorophore, increasing the number of probes to 50 loci spanning a genomic region of 500 kb on 11p15.4, to detect rearrangements causing &beta;-thalassemia or HPFH. Although slightly better results can be obtained when performing the PCR with the three sets of labeled universal primers separately, the ligation of all 50 probes was done in a single tube reaction. The fragment analysis was performed on a single sample of the three pooled PCR products per patient, which allowed the simultaneous analysis of 86 patient samples along with 10 normal controls in a 96 wells format fragment analysis run on the ABI3730.

The use of chemically synthesized oligonucleotides instead of cloning the half-probes into M13 vectors, as originally described for MLPA [11], allows cheap and rapid probe development, which increases the flexibility of MLPA for characterizing genomic rearrangements. Only 2 out of 85 probes (2%) were excluded from further calculations due to standard deviations higher than 12% when tested on a validation set of 12 wild type controls. The majority showed standard deviations between 0.05 and 0.08. Although these deviations seem significant, please note that due to the probe density rearrangements are mostly detected using a series of flanking probes (>2).

The ability to detect rearrangements in both regions was tested using positive controls, heterozygous for the 7 most common &alpha;-thalassemia deletions confirmed by gap-PCR, and for two less frequent mutations, Dutch I and -&alpha;7.9 confirmed by Southern blot analysis. By selecting 12 probes closely distributed along the 40 kb &alpha;-globin gene cluster, all of the common deletion types (except for the --FIL and --THAI ) could be distinguished from each other by MLPA. In our eyes, the simplicity, work-load and cost make MLPA a superior alternative to Southern blot analysis when a single technique is preferred for the detection of deletions causing &alpha;-thalassemia in a research setting. When desired, gap-PCR can be used for independent confirmation. Similarly, 6 positive controls were selected, based on the confirmation by different methods (Southern blotting and/or direct sequencing of break point fragments) and tested for the beta-cluster probe set. All of the probes expected to be deleted were confirmed in the heterozygotes tested.

In 19 and 20 samples large rearrangements involving the &alpha;- and &beta;-globin genes respectively could be excluded. Point mutations or micro-deletions affecting expression and located in between the probes, would not be picked up by MLPA. However, since iron levels were not known for some patients and anemia due to iron deficiency could easily be mistaken for &alpha;- or normal HbA2 &beta;-thalassemia, we believe that negative samples may fall into this category.

Polymorphisms in the genome, interfering with probe annealing and ligation of the two probe pairs, may cause the loss of probe signal leading to a false positive MLPA result [11,14,36]. During the screening of patient samples suspected for &alpha;-thalassemia one case showed repeatedly the deletion of a single probe 5a (in Figure 2a), named the (&alpha;&alpha;)ZW deletion found in an adopted child. This probe was selected in a highly conserved region of the HS-40, not containing the polymorphic sites known to be present in human populations [37]. Deletion of this regulatory element is expected to give a severe down regulation of &alpha;-gene expression of the affected chromosome. Even though nothing can be said about the extent of deletion, the fact that the HbH Inclusion Bodies test was positive and that no other rearrangements involving the &alpha;-genes were found, are strongly in favor of a deletion involving the HS-40. Whether or not this deletion, which is at maximum 30 kb in length, involves also HS-33 as found by Higgs et al. [38] needs further analysis. These types of deletions in human carriers may contribute to understand the mechanisms involved in regulation of downstream &alpha;-gene expression [39] and will be studied further.

In conclusion MLPA is an attractive alternative for FISH analysis for screening large deletions, for example in ATR-16 syndrome [9,10]. The tiling paths of cloned probes presently available for cytogenetic analysis of the 16p13.3 and 11p15.4 are shown in Figure 1 and 2. The distribution of synthetic probes coincides with the available cosmids, and allow a higher resolution of mapping than the available BAC or PAC probes. In contrast to in situ hybridization no laborious cell culture to generate metaphase spreads is necessary. MLPA can be performed directly on (stored) DNA samples.

MLPA uses standard technology only, i.e. hybridization, ligation, PCR and capillary electrophoresis. Since most diagnostic labs have these technologies operational, implementation of MLPA should be rather straight forward. The robustness, simplicity and intrinsic redundancy (probe density) of this approach, and additional specificity offered by the ligation step makes MLPA an attractive technique for the detection and characterization of copy number variaton (deletions/duplications) in any region of the genome, particularly for high resolution analysis, and those regions not amenable to analysis by array Comparative Genomic Hybridization (Array CGH) (Locke et al. 2004) [40].

'''The MLPA kits &alpha;-glopbinXS and &beta;-globinXS are supplied with all necessary buffers and enzymes by WWW.ServiceXS.com

Principle
MLPA stands for Multiplex Ligation-dependent Probe Amplification. The technique is used to detect unusual copy numbers of genomic sequences such as insertions or deletions. Basically MLPA is a method to make a DNA sample suitable for a multiplex PCR reaction in which up to 45 specific sequences are amplified simultaneously with the use of only one pair of PCR primers. The amplification products are then separated by electrophoresis (usually capillary electrophoresis) and insertions/deletions detected by comparison of relative peak heights with controls.

Ordinary multiplex PCR requires one pair of PCR primers for every fragment to be amplified. These PCR primers are present in large amounts during the PCR reaction and the presence of large numbers of different primers will cause problems. Ordinary multiplex PCR amplification cannot be easily used for relative quantification of the target sequences, as the efficiency of different primer pairs is not equal. Small differences in reaction conditions often result in large differences in the results obtained. MLPA reactions are more robust as all fragments are amplified with the use of only one pair of PCR primers.

In MLPA, a copy is made of each target sequence by hybridisation of two probes to each sequence. These probes consist of oligonucleotides that both hybridise to the sequence to be detected at sites immediately adjacent to each other. Each of these two probes contains one of the two sequences recognised by the PCR primers. The two parts of each probe can be ligated to each other by a specific ligase enzyme, but only when they are both hybridised at adjacent sites of the target sequence. These copy sequences are amplified in a multiplex PCR reaction with one primer pair.

Reagents
Most of the reagents are supplied in kit form from Service XS Holland.

Primer sequences
Number Name Sequence

540 IRD700 forward *GGCCGCGGGAATTCGATT

541 Reverse primer GCCGCGAATTCACTAGTG

* RD700 fluorescent labelled primer

Specimen type
Phenol Chloroform or Magnetic Bead extracted DNA

Standards
At least 3 normal DNA controls.

Internal quality Assurance
At least 2 DNA controls are included in each PCR run

The majority of reagents are supplied in kit form from ServiceXS.

Method
    Fill out the MLPA quantification, hybridisation and ligation log sheet (W059) with the standards/controls and patient samples to be tested.   The format of the run consists of:-   Lane 1-3: Three different normal samples (to be used as standards)   Lanes 4-5: Two control samples   Lanes 6-onwards: Test samples   Measure the DNA concentration of each sample in ng/ul using the eppendorf biophotometer.   Make a dilution (using sigma water) of 50ng/ul for each sample </li>  5ul of this dilution (i.e. 250ng of DNA) is used in the next step </li> </ol> </li>  <ol>  Program the Biometra UNO Thermo block for the first part of the MLPA reaction </li> <ol>  5 minutes at 98oC </li>  25oC pause </li>  2 minute at 95oC </li>  60oC pause </li>  15 minutes at 54oC </li>  5 minutes 98oC </li>  15oC pause </li>  The heated lid should be switched on at 105oC </li> </ol>  Label a 0.2ml thin-walled PCR for each sample and control and add 5ul of the DNA dilutions prepared previously. Put the tubes onto the programmed PCR machine and start the MLPA programme </li>  Meanwhile take out SALSA Probe mix (Grey cap) and MLPA buffer (yellow cap) to thaw. </li>  Make a master mix of 1.5ml SALSA Probe mix and 1.5ml MLPA buffer per reaction. Only use a pipette to mix the solution because using a centrifuge may cause the probes to precipitate out. </li>  Heat for 5mins at 98oC, wait until the PCR machine has reached 25oC before opening it. </li>  Add 3ml of the mix to each PCR tube whilst they are still at 25oC on the PCR block, mix again using a pipette. Close the lids and proceed to the next step of the PCR programme. </li> <li> Incubate for 2 minute at 95oC, then for 16 hours at 60oC (overnight) </li> </ol> </li> <li> <ol> <li> Make a master mix for the ligase reaction. This must be made less than 1 hour before used and stored on ice. </li> <li>Ligase-65 master mix (total volume 32ul per reaction) </li> <ol> <li> 3ml Ligase-65 buffer A (White cap) </li> <li> 3ml Ligase-65 buffer B (White cap) </li> <li> 1ml Ligase-65 (brown cap) </li> <li> 25ml Water </li> </ol> <li> Reduce the temperature of the PCR machine to 54oC by proceeding to the next step of the PCR programme. Whilst at 54oC add 32ml of Ligase-65 master mix to each sample whilst they are on the PCR block and mix using a pipette. This should be done as quickly as possible. Ensure the lids of the tubes are firmly closed before proceeding. </li> <li> Incubate the samples for 10-15 minutes at 54oC, then heat for 5 minutes at 98oC to deactivate the ligase enzyme. </li> <li> NB following ligase inactivation step samples can be stored for up to 1 week at 2-8oC or for longer periods at -20oC. </li> </ol> </li> <li>
 * Run set up and Quantification of DNA
 * Annealing and Hybridisation
 * Ligation reaction
 * PCR reaction

Do not use the primers supplied by ServiceXS as these are labelled with fluorescent dyes suitable for an ABI sequencer. Use primers 540 and 541 as described above. Also dNTPs which are not supplied with the kit need to be added. <ol> <li> Program the Biometra UNO Thermo block for the PCR part of the MLPA reaction </li> <ol> <li> 1 cycle 94oC for 5mins </li> <li> 33 cycles of 95oC for 50 sec </li> <li> 33 cycles of 60oC for 30 sec </li> <li> 33 cycles of 72oC for 1 min </li> <li> 1 cycle 72oC for 20min </li> <li> 15oC forever </li> </ol> <li> With heated lid switched on and set to 105 degrees C </li> <li> Label a second set of 0.2ml thin-walled PCR tubes, one for each sample and control and add 5ul of the ligated product prepared in step 3. </li> <li> Make the following master mix For 1 tube:- </li> <ol> <li> 10x SALSA PCR buffer (Red cap) 2ul <li> Sigma water 16.05ul <li> SALSA enzyme buffer (Blue cap) 1ul <li> Primer 540 (10pm/ul) 0.25ul <li> Primer 541 (10pm/ul) 0.25ul <li> dNTPs (25Mm) 0.2ul <li> SALSA Polymerase (Orange cap) 0.25ul </ol> <li> Add 20ul of master mix to each tube of ligated product and mix gently. Spin the tubes down, cap firmly and load on the thermal cycler. Start the programme as described above. </li> <li> NB The PCR products may be stored for 1 week at 2-8oC or indefinitely at -20oC. Fluorescent labels are light sensitive and should be stored in the dark. </li> </ol> </li> </ol>

Beckman Coulter Capillary Electrophoresis

 * 1) Make a master mix of 0.5ml of size standard 400 and 40ml of Beckman sample loading solution (per reaction)
 * 2) Label a Beckman CEQ 96 well plate with your run details. Transfer 40ml of the master mix to the number of wells required on the plate.
 * 3) Add 0.7ml of each MLPA PCR sample to it’s corresponding well and mix with a pipette
 * 4) Add a drop of mineral oil to each well
 * 5) Prepare a buffer plate
 * 6) Set up the run in the Beckman CEQ 8000 using create a new sample plate. Set the method for FRAG 6* and the analysis method for NEW ALPHA GREEN
 * 7) Load the sample and buffer plates and start the run.
 * 8) FRAG 6 method:
 * 9) Capillary temperature 50oC
 * 10) Denaturation 90oC for 90 seconds
 * 11) Injection time 2.0KV for 60 seconds
 * 12) Runtime 4.8KV for 60 minutes
 * 13) Analysis settings: Include peaks >3%, size standard-400, slope threshold 1.

Calculation of results
PCR products will be observed as peaks on an electrophoretogram. Print out the peak profile for each sample using the fragment analysis module on the Beckman CEQ 8000. There will be 20 peaks, C1 and C2 which represent control loci outside the alpha globin gene cluster. 18 other peaks which represent loci within the alpha globin gene cluster.

Analysis of the samples is done by visual examination of the peak profiles. This is done by comparing the peak height for each probe in the test sample with the peak height for that probe in the 3 normal controls.

In the normal samples 2 copies will be present for each probe target. In the test sample a deletion of one copy of the probe target sequence will usually be apparent by a reduction in relative peak height of 35-55%. A deletion of both copies of the probe target will result in the complete absence of the peak. Results, i.e. which peaks are dipped should be recorded on the MLPA PCR work sheet.

How to interpret results
The results are interpreted using the probe tables and alpha globin cluster maps supplied by ServiceXS. These are kept in a folder marked MLPA maps and information. Both work sheets for the run (WO59 and WO60) and all the peak profiles should be placed in a transparent folder and kept in the MLPA results file.