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MRC-Holland Description version 11; 17-07-2015 ® MLPA SALSA MLPA probemix P222-A2 LCA mix-2 Lot A2-0511. As compared to lot A1-0607, new control fragments (QDX2-fragments, 88 and 96 nt), one Xfragment (100 nt) and one Y-fragment (105 nt) have been added. Leber congenital amaurosis (LCA) is the most common inherited cause of blindness in childhood and is characterized by a severe retinal dystrophy before the age of one year. Eight genes have been identified that together account for approximately 45% of all LCA patients. This SALSA® MLPA® P222 LCA probemix-2 contains probes for 3 of these genes: GUCY2D, RDH12 and RPGRIP1. In addition, this probemix contains 2 probes for the CEP290 gene of which one detects the wild type sequence of the c.2991+1655A>G mutation. Mutations in the GUCY2D gene that encodes retinal guanylate cyclase-1 (retGC1) account for 10%-20% of all LCA cases. These mutations disrupt synthesis of cGMP in photoreceptor cells, a key second messenger required for function of these cells. The GUCY2D gene (20 exons) spans ~18 kb of genomic DNA and is located on chromosome 17p13.1, ~8 Mb from the p-telomere. The P222 probemix contains probes for 9 different GUCY2D exons. RDH12 encodes a photoreceptor-specific retinal dehydrogenase. Retinoids are indispensable light-sensitive elements of vision and also serve as essential modulators of cellular differentiation and proliferation in diverse cell types. RDH12 belongs to a family of dual-specificity retinol dehydrogenases that metabolize both all-trans- and cis-retinols (Haeseleer et al., J Biol Chem, 2002). The RDH12 gene (9 exons) spans ~33 kb of genomic DNA and is located on chromosome 14q24.1, ~68 Mb from the p-telomere. The P222 probemix contains one probe for exons 3 to 9. RPGRIP1 (RPGR-interacting protein 1) is a key component of cone and rod photoreceptor cells, where it interacts with RPGR (retinitis pigmentosa GTPase regulator). Mutations in RPGRIP1 have been detected in 4.5% of LCA patients. The RPGRIP1 gene (24 exons) spans ~63.3 kb of genomic DNA and is located on chromosome 14q11, ~21 Mb from the p-telomere. The P222 probemix contains probes for 18 different RPGRIP1 exons. The CEP290 protein is localized to the connecting cilium of photoreceptors. The gene is located at chromosome 12q21.33, ~88 Mb from the p-telomere and spans ~93 kb of genomic DNA. Its mutation c.2991+1655AG seem to be an important cause of LCA and may explain up to 21% of LCA cases. (den Hollander et al., Am J Hum Genet, 2006). The P222 probemix contains 2 probes for the CEP290 gene. One probe is specific for the wild type sequence of the c.2991+1655AG mutation and the other probe detects a sequence at a short distance from the mutation specific probe. This SALSA® MLPA® probemix is designed to detect deletions/duplications of one or more sequences of the aforementioned genes in a DNA sample. Heterozygous deletions of recognition sequences should give a 3550% reduced relative peak area of the amplification product of that probe. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak area, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA® MLPA® test. SALSA® MLPA® probemixes and reagents are sold by MRC-Holland 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® MLPA® test probemixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA® MLPA® 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). SALSA MLPA probemix P222 LCA mix-2 Page 1 of 6 MRC-Holland Description version 11; 17-07-2015 ® MLPA Related SALSA® MLPA® probemixes P221 LCA mix-1: Contains probes for the AIPL1, CRB1, CRX and RPE65 genes. More information Website : www.mlpa.com E-mail : [email protected] (information & technical questions); [email protected] (for orders) Mail : MRC-Holland bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands Data analysis The P222-A2 LCA mix-2 probemix contains 42 MLPA probes with amplification products between 124 and 427 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt, three DNA denaturation control fragments (D-fragments) at 88-92-96 nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this probemix can first be normalised intra-sample by dividing the peak area of each probe’s amplification product by the total area of only the reference probes in this probemix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised probe ratio in a sample by the average intra-normalised probe ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised 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 MRC-Holland, can be downloaded free of charge from our website www.mlpa.com. Many copy number alterations in healthy individuals are described in the database of genomic variants: http://projects.tcag.ca/variation. For example, a duplication of a complete gene might not be pathogenic, while a partial duplication or a deletion may result in disease. For some genes, certain in-frame deletions may result in a very mild, or no disease. Copy number changes of reference probes are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This probemix was developed by N. Laddach & J.P. Schouten at MRC-Holland. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the probemix designer could be made a co-author. Info/remarks/suggestions for improvement: [email protected]. SALSA MLPA probemix P222 LCA mix-2 Page 2 of 6 MRC-Holland ® MLPA Description version 11; 17-07-2015 Table 1. SALSA MLPA P222-A2 LCA mix-2 probemix Length (nt) 64-70-76-82 88-92-96 100 105 124 130 ~ 139 143 148 154± 159 166 172 177 184 190 195 ‡ 202 208 214 220 230 238 247 256 261 266 274 283 292 301 310 319 328 337 346 355 364 370 378 382 391 400 * 408 418 427 SALSA MLPA probe reference Chromosomal position GUCY2D RDH12 RPGRIP1 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation X-fragment: Specific for the X chromosome Y-fragment: Specific for the Y chromosome Reference probe 04235-L08770 8p22 GUCY2D probe 07036-L06647 Exon 3 RPGRIP1 probe 07023-L06634 Exon 10 RDH12 probe 08890-L09782 Exon 5 RPGRIP1 probe 07018-L06629 Exon 2 GUCY2D probe 07038-L06649 Exon 7 GUCY2D probe 07040-L06651 Exon 12 Reference probe 01588-L06028 17p13 RPGRIP1 probe 07032-L06643 Exon 22 RPGRIP1 probe 07021-L06632 Exon 6 RPGRIP1 probe 07024-L06635 Exon 11 Reference probe 03574-L02400 7q31 RPGRIP1 probe 07033-L08019 Exon 23 GUCY2D probe 07035-L06646 Exon 2 RPGRIP1 probe 07019-L06630 Exon 3 RPGRIP1 probe 07025-L06636 Exon 13 RPGRIP1 probe 07029-L06640 Exon 19 RDH12 probe 08889-L08851 Exon 4 RPGRIP1 probe 07030-L06641 Exon 20 GUCY2D probe 07037-L06648 Exon 5 GUCY2D probe 07039-L08018 Exon 10 RPGRIP1 probe 07031-L06642 Exon 21 Reference probe 00830-L00345 17q21 CEP290 probe 08887-L08849 GUCY2D probe 07041-L06652 Exon 14 RPGRIP1 probe 07017-L06628 Exon 1 RPGRIP1 probe 07022-L06633 Exon 8 RPGRIP1 probe 07028-L06639 Exon 18 RDH12 probe 08892-L08854 Exon 7 RPGRIP1 probe 07020-L06631 Exon 4 GUCY2D probe 07042-L06653 Exon 17 RPGRIP1 probe 07027-L06638 Exon 16 RPGRIP1 probe 07034-L06645 Exon 24 RDH12 probe 08893-L08855 Exon 8 RPGRIP1 probe 08141-L06637 Exon 14 Reference probe 05921-L05366 17q11 GUCY2D probe 07043-L06654 Exon 20 RDH12 probe 08891-L08853 Exon 6 CEP290 probe 08886-L08848 RDH12 probe 08894-L08856 Exon 9 RDH12 probe 08888-L08850 Exon 3 Reference probe 00680-L00121 7q34 CEP290 Intron 26 Intron 26 ~ This probe has been found duplicated in healthy individuals. Please disregard. ‡ More variable. This probe has a low signal and will be replaced in the next lot. * This probe detects the common allele of the intronic polymorphism c.2991+1655AG. The presence of the rare allele will result in a lower signal. ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Note The RDH12 and RPGRIP1 exon numbering has changed. From description version 06 onwards, we have adopted the NCBI exon numbering that is present in the latest NM_ sequences for the RDH12 and RPGRIP1 gene. This exon numbering might be different as compared to literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2. SALSA MLPA probemix P222 LCA mix-2 Page 3 of 6 MRC-Holland ® MLPA Description version 11; 17-07-2015 Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: [email protected]. Table 2. P222 probes arranged according to chromosomal location Table 2a. GUCY2D gene Length (nt) SALSA MLPA probe GUCY2D Exon start codon Ligation site NM_000180.3 75-77 (ex 2) Partial sequence (24 nt adjacent to ligation site) 202 130 ~ 247 07035-L06646 07036-L06647 07037-L06648 Exon 2 Exon 3 Exon 5 CAGCCTGCTCCG-TCTGTGTTCGCA TGCACTCGGTGC-TGCTGGGTGGCG CTCCTGTCACTG-TCCCTTCAGGAC 0.9 kb 3.2 kb 0.9 kb 154± 07038-L06649 Exon 7 CCTGGGCCACCT-AGCAGGTAGAGG 4.6 kb 256 159 283 337 382 07039-L08018 07040-L06651 07041-L06652 07042-L06653 07043-L06654 Exon Exon Exon Exon Exon 13 nt before ex 2 822-823 9 nt before ex 5 1 nt before ex 7 reverse 2103-2104 2411-2412 2688-2689 3154-3155 3483-3484 TAGTGGATGGCA-GATTCGTACTCA GCACCTGTCGAG-TGTATCCTCCTG GGACACCAGTGG-AGCCCGAGTACT CACTGTGGGGAT-TCTCCGTGCTCT TGGGGTGAACTG-CTCCTTGGCAGG 1.4 0.9 1.1 4.2 10 12 14 17 20 Distance to next probe kb kb kb kb stop codon 3384-3386 (ex 19) ~ This probe has been found duplicated in healthy individuals. Please disregard. ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. The NM_000180.3 sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. RDH12 gene Length (nt) SALSA MLPA probe 418 230 143 391 319 364 408 08888-L08850 08889-L08851 08890-L09782 08891-L08853 08892-L08854 08893-L08855 08894-L08856 RDH12 Exon start codon Exon Exon Exon Exon Exon Exon Exon 3 4 5 6 7 8 9 Ligation site NM_152443.2 325-327 (ex 3) Partial sequence (24 nt adjacent to ligation site) 255-256 420-421 581-582 80 nt before ex 6 29 nt before ex 7 74 nt before ex 8 1629-1630 GCCAGACCAGGA-ACCTGAGCCAGA GGAGTGTGTAGA-ACAAATGTGCAG AATCCGAGTGGA-TACAAAGAACTC TGAACAAAGGGA-AAGGGCAATTAT GTCATTCCACTT-TCAATCTTCCCT TCACTTGTGTAT-TTTGCTGCAGGA GCTGAGGCAAGA-AGAGCACCATCA Distance to next probe 1.9 0.7 0.8 1.0 2.2 5.1 kb kb kb kb kb kb stop codon 1273-1275 (ex 9) The NM_152443.2 sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete probe sequences are available on request: [email protected]. Please notify us on any mistakes: [email protected] SALSA MLPA probemix P222 LCA mix-2 Page 4 of 6 MRC-Holland ® MLPA Description version 11; 17-07-2015 Table 2c. RPGRIP1 gene Length (nt) SALSA MLPA probe RPGRIP1 Exon start codon Ligation site NM_020366.3 1-3 (ex 1) Partial sequence (24 nt adjacent to ligation site) 292 148 208 328 07017-L06628 07018-L06629 07019-L06630 07020-L06631 No probe 07021-L06632 No probe 07022-L06633 No probe 07023-L06634 07024-L06635 No probe 07025-L06636 08141-L06637 No probe 07027-L06638 No probe 07028-L06639 07029-L06640 07030-L06641 07031-L06642 07032-L06643 07033-L08019 07034-L06645 Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Exon 10 Exon 11 Exon 12 Exon 13 Exon 14 Exon 15 Exon 16 Exon 17 Exon 18 Exon 19 Exon 20 Exon 21 Exon 22 Exon 23 Exon 24 31-32 122-123 3 nt before exon 3 556-557 CTACATCAGGAG-ACTTGCCAGTTA CTTGAGCAGGAT-GAACCGGGAGGA TCTATTTGTCCA-CAGGCTGAGGAC CAAATGAAAACA-GAGGTGAAGTAG 6.7 6.2 1.6 5.2 4 nt before exon 6 GCGCCTTTCTCT-GCAGAGCTTCCA 4.2 kb 5.8 kb 995-996 GCTCAGGGCAGA-GCTGAAGGAAGA 1178-1179 1349-1350 TGACAGCTCCAG-TCAGCCCCACTG CAACATACTTCA-GAAGCATAAACA 2.3 kb 1.9 kb 1751-1752 1977-1978 AAGCCATGACCT-TCCAACATCTGG TATTCCTTCTAT-GACTTTGAAACC 2.8 kb 1.1 kb 2485-2486 TGTACCGCTTCT-TCACCTTTTCTG 2.5 kb 2925-2926 3131-3132 3273-3274 3379-3380 3599-3600 3654-3655 3783-3784 GAGGTTCCCATT-GAAGCTGGCCAG GAAGTTCTCAGA-GACTAACAGCTT TCTGAAGTCAGT-GAAGCACAAACT TCTCCCTGGCCT-TCTACCCAGAGG GCTGAATGGACA-AGATCCTGATCA CCTCTGGATGAA-GAAAAGAAAGAA ACCCCAATAGGA-AGGCTGAAGGTT 1.8 4.4 8.4 2.1 3.0 3.0 stop codon 3859-3861 (ex 24) 177 301 139 184 214 370 346 310 220 238 261 172 195 ‡ 355 Distance to next probe kb kb kb kb kb kb kb kb kb kb ‡ Low signal. This probe is less reliable and will be replaced in the next lot. The NM_020366.3 sequence is a reference standard in the NCBI RefSeqGene project. Table 2d. CEP290 gene Length (nt) SALSA MLPA probe CEP290 Exon start codon 400 * 08886-L08848 Intron 26 274 08887-L08849 Intron 26 stop codon Ligation site NM_025114.3 345-347 (ex 2) 1655 nt after exon 26 1791 nt after exon 26 reverse Partial sequence (24 nt adjacent to ligation site) TGTAATTGTGAA-TATCTCATACCT Distance to next probe 0.1 kb ACAGGGTAGGAT-TCATGTTTAGAA 7782-7784 (ex 54) * This probe detects the common allele of the intronic polymorphism c.2991+1655AG. The presence of the rare allele will result in a lower signal. The NM_025114.3 sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete probe sequences are available on request: [email protected]. Please notify us on any mistakes: [email protected] SALSA MLPA probemix P222 LCA mix-2 Page 5 of 6 MRC-Holland ® MLPA Description version 11; 17-07-2015 SALSA MLPA probemix P222-A2 LCA mix-2 sample picture 35000 188.78 121.25 259.45 213.09 30000 308.45 247.24 207.53 25000 96.22 20000 146.37 219.89 354.12 319.86 200.32 105.41 391.11 336.29 282.46 182.36 127.92 377.70 345.24 266.35 151.61 15000 299.71 273.89 255.32 289.79 137.14 90.99 85.67 229.72 237.23 175.97 170.35 165.55 326.03 407.10 417.73 369.26 382.36 141.50 363.19 426.69 157.41 D ye S ign al 10000 400.64 194.39 100.55 5000 0 100 150 200 250 Size (nt) 300 350 400 450 Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P222-A2 LCA mix-2 (lot A2-0511). Implemented Changes – compared to the previous product description version(s). Version 11 (48) - Electropherogram picture of the old buffer (introduced Dec. 2012) removed. Version 10 (48) - Warning added in Table 1 and Table 2, 154 nt probe 07038-L06649. Version 09 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 08 (48) - Minor textual changes. Version 07 (46) - Remark on RefSeqGene standard added below Table 2. - Minor textual changes. Version 06 (46) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Warning added in Table 1, 408 nt RDH12 probe 08894-L08856. - Various minor textual changes on page 1 and 2. - Exon numbering of the RDH12 and RPGRIP1 gene has been changed in Tables 1 and 2. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 05 (44) - Various minor textual changes on page 1. - Minor changes in the data analysis section on page 2. - Sentence “when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient” removed from data analysis section. - Tables have been numbered. - Various minor layout changes in Table 1 and 2. SALSA MLPA probemix P222 LCA mix-2 Page 6 of 6