SALSA MLPA probemix P200-A1 Human DNA Reference-1 Lot A1-1113, Lot A1-0410, A1-0209, A1-0408

Transcription

1 SALSA MLPA probemix P200-A1 Human DNA Reference-1 Lot A1-1113, Lot A1-0410, A1-0209, A The P200-A1 Human DNA Reference-1 probemix contains reference probes and control fragments specific for unique human DNA sequences. The P200 probemix offers a collection of thoroughly-tested MLPA reference probes and control fragments to which home-made synthetic MLPA probes can be added. Not only do these control fragments and reference probes provide extra assurance about the quality of selfmade probes and of the MLPA reaction performed, they also facilitate the data analysis of your synthetic probemix and maximise the number of synthetic probes you can include. Next to the P200, also offers the P300 Human DNA Reference-2 probemix. While the P200-A1 leaves the entire range from nt open for the inclusion of synthetic probes, the P300-A1 probemix has reference probes distributed over the whole size range of the probemix. This is especially advantageous for correction of the signal sloping of MLPA amplification products. Signal sloping is the effect that longer probes generate a lower peak height/area on the electropherogram than shorter probes; the exact amount of signal sloping differs between sequencer types and can differ between different samples. The use of P200 is recommended when a large number of synthetic probes are used, preferably targeting sequences on different chromosomes. Results can be difficult to interpret when P200 is used in combination with a small number of probes all targeting the same gene or chromosomal region, and with lengths much smaller than the reference probes. In samples with sloping, it may seem in such cases that all sequences targeted by the synthetic probes are deleted or duplicated. The use of P300 is recommended when a smaller number of synthetic probes is used. Guidelines on designing synthetic MLPA probes and the necessary RAW program are available on our website Unfortunately, we cannot offer additional support on probe design. Please read our synthetic probe design protocol carefully before you start designing. This P200-A1 Human DNA Reference-1 probemix contains 11 reference probes. In addition, the Q- fragments, one D-fragment, one chromosome Y-specific and one chromosome X-specific control probe are included. The Q-fragments give a warning when insufficient amounts of sample DNA are used. The D- fragment gives a warning when the sample DNA was not completely denatured. Method For each synthetic MLPA probe oligonucleotide, a 1 µm dilution in TE (10 mm Tris-HCl ph 8.0; 1 mm EDTA) should be made. E.g. dissolve 25 nmol oligo in 250 µl (= 100 µm) and then dilute 10 µl of this stock to 1 ml. Store the stock and 1 µm dilution at -20 o C. Make your synthetic probemix by combining 0.8 µl of each 1 µm oligo solution in a final volume of 200 µl TE. Store at -20 o C. For each MLPA reaction, use 1 µl P µl synthetic probe mix µl MLPA buffer. Always mix thawed oligonucleotide solutions before pipetting! SALSA probemixes and reagents are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in diagnostic procedures. Purchase of the SALSA test probemixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA probemix and reagents requires a thermocycler with heated lid and sequence type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA technique has been first described in Nucleic Acid Research 30, e57 (2002). More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA probemix P200 Human DNA Reference-1 Page 1 of 5

2 Related products P300 Human DNA Reference-2: contains control fragments and reference probes at 92, 109, 129, 148 and from 173 to 250 nt. Data analysis The P200-A1 Human DNA Reference-1 contains 14 MLPA probes with amplification products between 173 and 250 nt, including one D-fragment, one chromosome Y-specific and one chromosome X-specific control. In addition, it contains 4 DNA Quantity fragments (Q-fragments) at nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by the combination of your synthetic probemix + this probemix can be intra-normalized by dividing the peak height of each probe s amplification product by the total peak heights of only the reference probes in this probemix (block normalisation). Secondly, normalisation can be achieved by dividing this intranormalized probe ratio in a sample by the average intra-normalized probe ratio of all reference samples. Please note that this type of normalisation assumes that no changes occured in the genomic regions targeted by the reference probes. Data normalisation should be performed within one experiment. Only samples purified by the same method should be compared. Confirmation of most exons deletions and amplifications can be done by e.g. Southern blotting, long range PCR, qpcr, FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website Methylation-specific MLPA This probemix can also be used in combination with your home-made MS-MLPA probes, as the 184 nt probes contains a HhaI restriction site and can be used as an internal control for complete HhaI digestion. Upon proper HhaI digestion, the 184 nt probe signal will be almost gone. More information on methylation quantification by MS-MLPA can be found on Synthetic probe design Guidelines on how to design synthetic MLPA probes and the necessary RAW program are available on our website Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P200 Human DNA Reference-1 Page 2 of 5

3 Table 1. SALSA MLPA P200-A1 Human DNA Reference-1 Length (nt) SALSA MLPA probe Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA Chromosomal position other reference Hg18 * 173 Reference probe L q Mb 178 Reference probe L q Mb 184 # D-probe L08987: Low signal indicates incomplete denaturation 14q Mb 190 Reference probe L p Mb 196 Reference probe L q Mb 202 Reference probe L p Mb 208 Chromosome X probe L04391 Xq Mb 214 Reference probe L p Mb 220 Reference probe L q Mb 226 Reference probe L q Mb 233 Reference probe L q Mb 240 Chromosome Y probe L00464 Yq Mb 244 Reference probe L p Mb 250 Reference probe L p Mb * Distance to P-telomere. # When used for methylation quantification (MS-MLPA), this denaturation warning probe can also be used as digestion control probe (warning for insufficient HhaI digestion). Upon digestion of the probe-sample hybrids with HhaI, this probe should not give a signal. Note: The identity of the genes detected by the reference probes and complete probe sequences are available on request: info@mlpa.com. SALSA probemix P200 Human DNA Reference-1 Page 3 of 5

4 SALSA MLPA probemix P200-A1 Human DNA Reference-1 sample pictures D ye S ign al Size (nt) Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P200-A1 Human DNA Reference-1 (lot A1-1113) D ye S ign al Size (nt) Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human female control DNA analysed with SALSA MLPA probemix P200-A1 Human DNA Reference-1 (lot A1-1113). SALSA probemix P200 Human DNA Reference-1 Page 4 of 5

5 Implemented Changes compared to the previous product description version. Version 13 (53) - Product description adapted to a new lot (lot number added, new picture included). - Various minor textual changes Version 12 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 11 (48) - Textual changes on page 1. Version 10 (48) - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Various minor textual changes. Version 09 (45) - Product description adapted to a new lot (lot number added, new pictures included). - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section. - Sentence Complete probe sequences are available on request has been added on page 3. Version 08 - ph adjusted to Ph=8.0 so that it corresponds with the synthetic probe design protocol. Version 07 - Extended name has been changed from Reference-1 into Human DNA Reference-1. - Various minor textual changes on page 1. - Various minor layout changes. SALSA probemix P200 Human DNA Reference-1 Page 5 of 5