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aThe primers of the exons marked with an * were designed for this assay, the others are as in Kitada et al. (1). Forward primers are 5'-fluorescently labeled (with the same fluorchrome or see Note 3).

aThe primers of the exons marked with an * were designed for this assay, the others are as in Kitada et al. (1). Forward primers are 5'-fluorescently labeled (with the same fluorchrome or see Note 3).

2.2. Polyacrylamide Gel Electrophoresis

1. Prepare stocks of 5% denaturing polyacrylamide gels using Page-Plus 40% (Amersham) according to the manufacturer's recommendations. Keep 35-mL aliquots at 4°C and use within 4 wk.

2. An automated sequencer (e.g., ABIPRISM 377 upgraded for 96 wells by Applied Biosystems, see Note 3).

3. Internal size standard (TAMRA-500 and TAMRA-500 XL by Applied Biosystems) and its blue loading buffer.

4. Deionized formamide (Amersham).

2.3. Interpretation of Data

1. Fragment analysis software (e.g., Genescan 3.1 and Genotyper 1.1.1 software by Applied

Biosystems).

2. Calculation software (e.g., Excel 97 by Microsoft).

3. Methods

3.1. Multiplex PCR

1. Set up premixes (13 ||L per reaction) corresponding to the first 3 exon combinations (for combination 4, see Note 4) by adding 2.5 ||L of 10X buffer (final concentration 1X), 1.5 ||L of 50 mM MgCl2 (final concentration 3 mM), the appropriate amount of each primer (between 0.3 and 2.5 ||L, final concentration between 0.4 and 2.0 ||M, see Note 5), 0.2 ||L of 25 mM dNTP-Mix (final concentration 0.2 mM per dNTP) and complete with H2O up to 13 ||l per reaction. Prepare enough premix for duplicate (or more) reactions for each case.

2. Spot 2 ||L of the DNA (= 40 ng) on the wall of a 96-well PCR-plate. This is an easy way to control which well contains DNA (see Note 6).

3. Add 13 ||L of the appropriate premix to the well (see Note 7). Close the wells with lid strips, vortex briefly, and spin down the contents in a centrifuge equipped for PCR plates.

4. Prepare for a semi-hotstart (see Note 8) by heating the PCR apparatus to 94°C. Open the wells carefully, add 10 |L of Taq solution (2 U/10 |L water kept on ice) using a distribution pipet, close the wells with new strips, vortex briefly, tap the plate on the lab bench to collect all liquid at the bottom of the wells, and place it on the preheated PCR block.

5. Use the following cycling conditions in "tube simulation mode" for combinations 1-3: 94°C for 5 min (initial denaturation), 23 cycles of 94°C for 30 s, 53°C for 45 s and 68°C for 2.5 min, 68°C for 5 min (final extension). For combination 4, see Note 4.

3.2. Polyacrylamide Gel Electrophoresis

1. Prepare the sequencing gel by adding ammonium persulfate and TEMED, following the manufacturer's recommendations, to an aliquot of the acrylamide.

2. Prepare the loading buffer by pipetting together a premix containing: 0.3 |L of TAMRA-500 XL, 0.6 ||L of loading blue, and 3 |L of formamide (for sequencer with 36 lanes; see Note 3) per sample.

3. Pipet 3.5 ||L aliquots of this loading premix into the wells or tubes used to prepare the samples for loading.

4. Add 2-2.5 |L of the PCR products to the premix. Close the tubes, centrifuge briefly, vortex, and centrifuge again (for sequencers with 36 lanes; see Note 3).

Fig. 1. Four exon combinations used in multiplex PCRs. Representative electrophoregrams obtained after multiplex PCR with HEX-labeled forward primers and separation on 5% denaturing gels on an automated Sequencer (ABI PRISM 377). Peak length (indicated in bp above each electrophoregram) and peak height (indicated below each electrophoregram) are given as calculated by Genotyper 1.1.1 software (Applied Biosystems). Exons are numbered below each peak. Please note that two peak heights are given for double peaks the sizes of which have to be added (see Note 9). *, unspecific peak.

Fig. 1. Four exon combinations used in multiplex PCRs. Representative electrophoregrams obtained after multiplex PCR with HEX-labeled forward primers and separation on 5% denaturing gels on an automated Sequencer (ABI PRISM 377). Peak length (indicated in bp above each electrophoregram) and peak height (indicated below each electrophoregram) are given as calculated by Genotyper 1.1.1 software (Applied Biosystems). Exons are numbered below each peak. Please note that two peak heights are given for double peaks the sizes of which have to be added (see Note 9). *, unspecific peak.

Representative examples of electrophoregrams are given in Fig. 1. However, other peak patterns are also possible, as long as all exons amplify exponentially (see Subheading 3.4.). Exon 8 was amplified in comb 1 and comb 2, so that the same positive control (heterozygously deleted for exons 8 and 9) could be used in all three combinations.

2. More recently, we added a fourth exon combination: Ex1 (94 bp) + Ex3iDos (138 bp) in order to co-amplify exon 1. This is difficult, since the 5'- untranslated region of the parkin gene (containing the forward primer binding site for exon 1) is very GC-rich. We obtained the best results with the primers mentioned in Table 1 for exon 1 and exon 3iDos. However, a nonspecific peak is also obtained (see Fig. 1) that co-amplifies exponentially with the two exons (for PCR conditions; see Note 8). Since all known exon 3 deletions were detected with this combination, we assume that exon 1 deletions should also have been detected. However, we have not yet detected an exon 1 deletion in the patients studied.

3. In order to save space on the sequencing gel (e.g., with a 36 lanes sequencer), the exons of the first 3 combinations can be labeled with 3 different fluorchromes (e.g., comb 1 with TET, comb 2 with FAM, and comb 3 with HEX). Aliquots of the PCRs are then pooled in the loading buffer. Under these conditions, we obtained good results with 1 |L of comb 1, 1.5 |L of comb 2 and 3 |L of comb 3, added to 1.5 |L of TAMRA-500, 3 |L of formamide and 0.7 |L of loading blue. Combination 4 is run separately, since no other fluorchrome is available.

4. For multiplex combination 4, the best results were obtained with the following PCR protocol: in a final volume of 25 |L, 40 ng of DNA, 10% DMSO, 1.5 |M of each primer, 0.2 mM of each dNTP and 2 U of Taq polymerase. The cycling protocol consisted of 94°C for 4 min followed by 22 cycles of 94°C for 30 s, 53°C for 30 s and 68°C for 45 s. Final elongation was performed at 68°C for 10 m.

5. Concentrations of the primers varied from 0.40 |M to 2 |M. The exact primer concentrations have to be determined experimentally because they vary among batches.

In order to obtain approximately equal peak heights for the exons in each multiplex PCR combination and to be in the exponential phase for each exon, we followed in part the recommendations of Henegariou et al. (8), in particular: lowering the annealing temperature to 53 °C and extension temperature to 68°C, increasing the MgCl2 concentration to 3 |M and the extension time to 2.5 min (see Note 4 for combination 4). In addition, the primer concentrations have to be adjusted. We recommend starting with 0.8 |M for each primer and adapting the concentration according to the resulting peak height (i.e., primer concentrations have to be decreased for exons that amplify well and increased for exons that amplify less well).

6. In addition to the cases, at least one normal control and a negative control (without template DNA) should be run in parallel. We also included a positive control with known heterozygous deletions of exons 8 and 9 in order to control for false results due to variation in the premix. However, if no positive control with known exon rearrangements is available, the dosage assay can still be considered to be valid as long as PCR amplification is exponential.

7. In order to facilitate mixing of the reagents, pipet the premix onto the drop of DNA (e.g., with a Gilson Distriman with Distritips) but avoid touching the DNA to prevent carryover contamination.

8. If the PCR premix is kept on ice or if a Taq Polymerase with automated hot-start is used, the TaqPolymerase could also probably be added directly to the premix, although we have not done this.

9. Peak labeling programs can determine peak length (size of the PCR product), peak height, and peak area. Peak area would seem to be the most precise reflection of the amount of PCR product, but often the algorithms used only approximate the area. We have had more consistent results using peak height. It is important to sum the peak heights when a PCR product forms a double peak with 1 bp difference, caused by the addition of an A to the PCR products by the polymerase (see examples in Figs. 1-3). Genotyper does not always distinguish double peaks, so vigilence is necessary.

10. The interpretation of the data is based on the relative peak heights obtained within a given multiplex reaction. The method is therefore only semi-quantitative and does not give absolute values that would be affected by the amount and quality of template DNA as well as slight variations in the premix. In order to establish the peak height patterns for cases and controls, the ratios of the peak heights of each exon to every other exon in a given exon combination are calculated (see Tables 2 and 3). The average ratio of the duplicate/triplicate/quadruplicate reactions are then calculated and normalized with respect to the

Fig 2. Compound heterozygous deletions of exon 2 and 3. Representative electro-phoregrams of the cosegregation of two different exon deletions in a family compared to a control. Peak labeling is as in Fig. 1. For each case, one of the triplicate PCRs is shown. However, in Table 2, all three PCRs are used for the NR calculation. Circles, women; squares, men; filled symbols, patients; het, heterozygous; del, deletion.

control(s). The normalized ratios (NRs) obtained in different experiments (e.g., if experiments are performed several times with different batches of DNA) are therefore comparable and mean normalized ratios (MNRs) can be calculated.

In practice, a template "Excel" sheet should be established containing all the calculation instructions described below, so that only the crude peak heights need to be entered. Two examples are given in Tables 2 and 3, that correspond to the electrophoregrams in Figs. 2 and 3. In the left part of the table, the peak heights for each case are entered, e.g., the duplicate PCRs of controls 1 to 3 and the quadruplicate PCRs of the cases A and B (Table 3). In the right part of the table, next to the peak heights, the program calculates the peak height ratios as indicated at the top of the tables. Below, the program calculates the mean values (in bold) and the standard deviation of the ratios, respectively. Rearrangements are deduced from the division of the mean peak height ratios of all available controls (i.e., one control in triplicate in Table 2 and three controls in duplicate in Table 3) by the mean peak height ratios of the case. This results in the NRs for each exon peak height ratio (shown in bold italics for normal values or bold underlined for pathological values in the table). The NRs have a value of approx 1 (i.e., 0.8 to 1.2) for nonrearranged exons (2 copies of an exon). Values of 0.6 or less are interpreted as one copy of an exon (i.e., heterozygous deletion), values of 1.3 to 1.7 as 3 copies (i.e., heterozygous duplication), values of 1.8 to 2.3 as 4 copies (i.e., homozygous duplication or heterozygous triplication)

Fig 3. Homozygous duplication of exon 3. Representative electrophoregrams of the cosegregation of an exon duplication in a family compared to 3 controls. In addition, a positive control with known heterozygous deletions of exons 8 and 9 was run in parallel. All values of the duplicate or quadruplicate PCRs are shown in table 3. Peak labeling and symbols are as in Figs.1 and 2. Het, heterozygous; hom, homozygous; del, deletion; dupl, duplication.

and values above 2.6 as indication for 6 copies (i.e., homozygous triplication) (see Fig. 4). Please note that the NR values given above apply only if the exon under investigation is the dividing value in the exon ratio, i.e., C328/3i. If the ratio is Ex3i/12 (e.g., case C in Table 2), the NR becomes approx 2 (1/0.5). An exon rearrangement is confirmed only if all of the ratios concerning the exon are abnormal (see tables). If the results are ambiguous, the PCR should be repeated, if possible with a new DNA sample and a larger number of replicates (e.g., up to quadruplicate). In addition, several control cases can be included to obtain mean values for the control ratios.

11. Reproducibility. As seen from the tables, standard deviations are usually about 10% of the mean, and rarely exceed 20%, for both the crude exon ratios calculated from the PCR replicates and the mean NRs from several different experiments (data not shown).

12. Search for homozygous exon deletions. In addition to the detection of heterozygous exon deletions, the multiplex PCR assay also detects homozygous exon deletions that are obvious, since one or several peaks are missing.

Table 2

Calculation of Exon Ratios, SD, and Normalized Ratios Corresponding to Fig. 2"

Case

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