Genetic testing of Tay-Sachs disease by PCR and Restriction Digest ESC102-PRA0103 Submitted to: Elizabeth Berndl 18 February, 2009 Submitted by: Laila Hulbert (996625077) Maria Yancheva (996742173)
Scientific and Medical Justification Tay-Sachs disease is a progressive neurodegenerative disorder, which causes large quantities of the GM2 ganglioside fatty acid to accumulate in the nerve cells of the brain [1]. The presence of gangliosides in the retinal cells results in the appearance of a red spot in the retinae of patients with Tay-Sachs disease. Other consequences caused by the excess presence of gangliosides in the nerve cells are a loss of mental and physical abilities in infants, leading to blindness and deafness [2]. The atrophy of muscles causes paralysis and eventual death at an early age. In its classic form, the disease is found in infants and is typically fatal by the age of 2 or 3. Tay-Sachs is a genetic disorder caused by a mutation in the hexosaminidase A (HEXA) gene [1], and is inherited in an autosomal recessive pattern [2]. There are a number of genetic mutations which can cause the disease. The Arg170Gln mutation was found to be one of the wide-spread mutations causing Tay-Sachs among the Moroccan Jewish population [1]. There is currently no cure for the disease, and infantile Tay-Sachs disease results in death by the age of 5 at best. The methods of treatment currently being researched include enzyme replacement therapy which aims to replace a missing enzyme in patients; gene therapy which aims to replace defective genes through the brain; and substrate reduction therapy which aims to control the metabolism of GM2 gangliosides in the brain [2]. This proposal describes a diagnostic which tests for the Arg170Gln mutation caused by the substitution of glutamine in place of arginine-170, resulting in a G-to-A change at nucleotide 509 [1]. See Appendix A for transcript and genomic region. The primers used to amplify the region are as follows: (see Appendix B for primer design details) Tay-Sachs F: CTTGAGGCCACACTGCTTTGAA Tay-Sachs R: TGTATCCCTGGAAACAGCCTGA The restriction enzyme HpaII will be used to cleave these sites (see Appendix C for cleave sites). The following bands will be produced. Homozygous Wild Type: 346, 614 Heterozygous: 346, 614, 960 Homozygous Mutant: 960 Protocol (Modified from Labs 2 & 3) Step One: Obtaining DNA See Lab 2, step1. Step Two: PCR Amplification of DNA See Lab 2, step 2, except: After 3, each group member transfers the remaining Instagene liquid (without beads) to a 1.5 ml tube, labeled with her name on a removable label. The TA removes the label with the name and replaces it with a number, and selects three random samples to test. Transfer 20 μl of master mix into each PCR tube, mix by pipetting up and down two to three times. Avoid bubbles. Cap the tubes and keep them on ice. Continue from step 6 in protocol. Step Three: Restriction Digest and Electrophoresis See Lab 3, except: Add 10 μl PCR product and 10 μl HpaII master mix to microtubes, and incubate at 37C for 1 hour (do this before preparing gel). Continue from 1.
Concentration of agarose gel should be 2% (from RFP) After incubation, add 5 μl loading dye to one undigested sample and to all digested samples. Continue from step 6. Step Four: Staining with Sybr Green See Lab 3.
References [1] OMIM. (2009). Tay-Sachs Disease. Retrieved February 17, 2009, from http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=272800. [2] Wikipedia. (n.d.). Tay-Sachs Disease. Retrieved February 17, 2009, from http://en.wikipedia.org/wiki/tay-sachs.
Appendix A Wild Type and Tay-Sachs cell variants of the HEXA gene from Ensembl Wild Type Transcript: 961 ACATTCTTTATCAACAAGACTGAGATTGAGGACTTTCCCCGCTTTCCTCACCGGGGCTTG 153 -T--F--F--I--N--K--T--E--I--E--D--F--P--R--F--P--H--R--G--L- Mutant Transcript: 961 ACATTCTTTATCAACAAGACTGAGATTGAGGACTTTCCCCGCTTTCCTCACCaGGGCTTG 153 -T--F--F--I--N--K--T--E--I--E--D--F--P--R--F--P--H--R--G--L- Wild Type Genomic Region: 22861 ACCCAGGCTCCTTGGAAACCCAAGCTTATGATCACTTTCTCACAATTTATTCTGGGACCC 22920 22921 CTTTTCTCAGCTCTCTTAGCTCCTGATAGGGGTTATCCATGAAAAGTCCAACTCTTGCAG 22980 22981 GCTGAGTTGTCTCTGTGTCTGTCCTCATGAGTATATGTCTTGGCCTCTCTTCTCAATATT 23040 23041 CTTGTGAAGGCACAGGAAATATAGATAAGAGGTATGTGGGTTTTGAAGGTTAGTATGTTG 23100 23101 TAAGGAAGGGCACTAAAACCCTTACTCTGACATCTCCCAAAATGAAATACAGAAGTAAAG 23160 23161 ATGGATAGTGGCTTCCTAATATCCCCTTTTCATCAGTGTTAAAAATCGTCAAGCCTACAC 23220 23221 TACATCCATTTTCCCTGTGTACCCAAATGTTATGCTCTGCTACATTGAGAACCTTCCAAA 23280 23281 TAGAGACATCTCTTTTTCCACTCCCTGAGTCTTTACTCTGCCTCTCTACATCTTCCTAGG 23340 23341 TCTGGAGACTTTTAGCCAGCTTGTTTGGAAATCTGCTGAGGGCACAGTAAGTGTGGACCC 23400 23401 TCAAAAAGGGCTCCTTTTTCATCTCTGGGGTTGGATCCCAATATTGAGAATCCTAGAAAA 23460 23461 ATCTTGTTTTGCCTTAGGATCCTGGTGGCTTGAGATAGCCCCAGTAGAAATGGAAGAGGA 23520 23521 GATGTGAGTGGAAAACTGGTTTGGTAGATTTGGTCTGTTTGGAATCACTGTAATTTGGTC 23580 23581 CCCCTGAGTCATCATTTAGTAATGACCTTGAGGCCACACTGCTTTGAAGAAAATAAACTA 23640 23641 TGTTAACAGGAAAATCCAGAATTTGTAGGCTGAGTGGTGTTTGGGACAGTTGTATTAGGC 23700 23701 TGGCGTTGGACAATTGCTCTCTGGGAAGGTGGCTGCTTATGGTCATTTATAGAGGTGGGG 23760 23761 AGCATTTAGGGAGAGAGCGACCCTGAGAACAGTCACAGATTGGCTTTTTAAGAATCCTGG 23820 23821 GAGAGTTGTCTTCATCTCCCTGTGCCCCCATAGTAAGCTTCCTTTGAAACTGACATGTTC 23880 23881 AATGTTTGTTCTGCACAGTTCTTTATCAACAAGACTGAGATTGAGGACTTTCCCCGCTTT 23940 23941 CCTCACCGGGGCTTGCTGTTGGATACATCTCGCCATTACCTGCCACTCTCTAGCATCCTG 24000 24001 GACACTCTGGTAACCAGGCCTTAAGGCCTTCTTCACACTTAAGAGTTCTAGGGTGATGGA 24060 24061 GCAACGGACAGACCAAGTTCCAAATTCTTAAAGAGTTAGAGAGGGAATGTATGGACTGGG 24120 24121 TGTGGTGGCTCATGCCTGTAATCCCAACACTTTGGGAGGCCGAGGTGGGCGGATCACCTG 24180 24181 AAGTCAGAAGTTCAAGGCCAGCCTGGCCAACATGGTGAAACTCTGTCTCTACTAAAAATA 24240 24241 CAAAAATTAGCTGGGCATGGTGGTGCATGCTTGTAATCCCAGCTACCTGGGAGGCTGAGG 24300 24301 CAGGAGAATCGCATGAACCCAGGAGGCAGAGGTTGCAGTGGGTGGAGATCACGCCACTGC 24360 24361 ACTCCAGCCTGGGTGAAAGAGTGAGACGCTGTCTCAAAAAAAAAAAAAAAAATTGTATGT 24420 24421 AATTCTATGAGCTCCTGGGAAAAATGTAGAAGATGAGCTGCTCCAAGACTCAAAAAATAA 24480 24481 CAGAGTGACTTAAATCTACCTTACAGGCTACTGTAGATCAATGTTCAGGCTGTTTCCAGG 24540 24541 GATACAAATGTTTAGGGGAATTGGCTTCATTGGCAGAGCTAACTATATTTGTTTTTGAGA 24600 24601 CAGCGTCTCAATTTTTTTTTTTTTTTTTTGTATTTTTTGTAGAGCTGGGGTTTCGTCATG 24660 24661 TTACCCAGGCTGGTCTTGAACTCCTGGGCTCAAGCAATCTGCCCACCTTGGCCTGCCAGA 24720 24721 GTGGTAGAATTACAGGCATGAGTCACTACACCTGGCCGGCAGAGCTAACTTTAGTTAGAA 24780 24781 CAAGGTCATAGGAGAGAGACGTTCAAAGCCTTAACTTGGGGCCAGGTGCAGTGGCTCACA 24840 24841 CCTGTAATCCTAGCACTCTGGGAGGCCAAGGCGGGTGGATTGCCCGAGCTCAGGAGTTTG 24900 24901 AGACTAACCTGGCCAAGACGGTGAAACCCCATCTCTACTGAAAATACAAAAAATTAGCCA 24960 24961 GGCGCGATGGCGCAAACCTGTAGTCCCAGCTACTCAGGAGTCTGTGATGGGAGAATCACC 25020 25021 TGAGCCTGGGGAGATCAAGGATGCATGAACTGTGATTGCACCACTGCACTCCAGCCTGGA 25080 Mutant Genomic Region: 22861 ACCCAGGCTCCTTGGAAACCCAAGCTTATGATCACTTTCTCACAATTTATTCTGGGACCC 22920 22921 CTTTTCTCAGCTCTCTTAGCTCCTGATAGGGGTTATCCATGAAAAGTCCAACTCTTGCAG 22980 22981 GCTGAGTTGTCTCTGTGTCTGTCCTCATGAGTATATGTCTTGGCCTCTCTTCTCAATATT 23040 23041 CTTGTGAAGGCACAGGAAATATAGATAAGAGGTATGTGGGTTTTGAAGGTTAGTATGTTG 23100
23101 TAAGGAAGGGCACTAAAACCCTTACTCTGACATCTCCCAAAATGAAATACAGAAGTAAAG 23160 23161 ATGGATAGTGGCTTCCTAATATCCCCTTTTCATCAGTGTTAAAAATCGTCAAGCCTACAC 23220 23221 TACATCCATTTTCCCTGTGTACCCAAATGTTATGCTCTGCTACATTGAGAACCTTCCAAA 23280 23281 TAGAGACATCTCTTTTTCCACTCCCTGAGTCTTTACTCTGCCTCTCTACATCTTCCTAGG 23340 23341 TCTGGAGACTTTTAGCCAGCTTGTTTGGAAATCTGCTGAGGGCACAGTAAGTGTGGACCC 23400 23401 TCAAAAAGGGCTCCTTTTTCATCTCTGGGGTTGGATCCCAATATTGAGAATCCTAGAAAA 23460 23461 ATCTTGTTTTGCCTTAGGATCCTGGTGGCTTGAGATAGCCCCAGTAGAAATGGAAGAGGA 23520 23521 GATGTGAGTGGAAAACTGGTTTGGTAGATTTGGTCTGTTTGGAATCACTGTAATTTGGTC 23580 23581 CCCCTGAGTCATCATTTAGTAATGACCTTGAGGCCACACTGCTTTGAAGAAAATAAACTA 23640 23641 TGTTAACAGGAAAATCCAGAATTTGTAGGCTGAGTGGTGTTTGGGACAGTTGTATTAGGC 23700 23701 TGGCGTTGGACAATTGCTCTCTGGGAAGGTGGCTGCTTATGGTCATTTATAGAGGTGGGG 23760 23761 AGCATTTAGGGAGAGAGCGACCCTGAGAACAGTCACAGATTGGCTTTTTAAGAATCCTGG 23820 23821 GAGAGTTGTCTTCATCTCCCTGTGCCCCCATAGTAAGCTTCCTTTGAAACTGACATGTTC 23880 23881 AATGTTTGTTCTGCACAGTTCTTTATCAACAAGACTGAGATTGAGGACTTTCCCCGCTTT 23940 23941 CCTCACCaGGGCTTGCTGTTGGATACATCTCGCCATTACCTGCCACTCTCTAGCATCCTG 24000 24001 GACACTCTGGTAACCAGGCCTTAAGGCCTTCTTCACACTTAAGAGTTCTAGGGTGATGGA 24060 24061 GCAACGGACAGACCAAGTTCCAAATTCTTAAAGAGTTAGAGAGGGAATGTATGGACTGGG 24120 24121 TGTGGTGGCTCATGCCTGTAATCCCAACACTTTGGGAGGCCGAGGTGGGCGGATCACCTG 24180 24181 AAGTCAGAAGTTCAAGGCCAGCCTGGCCAACATGGTGAAACTCTGTCTCTACTAAAAATA 24240 24241 CAAAAATTAGCTGGGCATGGTGGTGCATGCTTGTAATCCCAGCTACCTGGGAGGCTGAGG 24300 24301 CAGGAGAATCGCATGAACCCAGGAGGCAGAGGTTGCAGTGGGTGGAGATCACGCCACTGC 24360 24361 ACTCCAGCCTGGGTGAAAGAGTGAGACGCTGTCTCAAAAAAAAAAAAAAAAATTGTATGT 24420 24421 AATTCTATGAGCTCCTGGGAAAAATGTAGAAGATGAGCTGCTCCAAGACTCAAAAAATAA 24480 24481 CAGAGTGACTTAAATCTACCTTACAGGCTACTGTAGATCAATGTTCAGGCTGTTTCCAGG 24540 24541 GATACAAATGTTTAGGGGAATTGGCTTCATTGGCAGAGCTAACTATATTTGTTTTTGAGA 24600 24601 CAGCGTCTCAATTTTTTTTTTTTTTTTTTGTATTTTTTGTAGAGCTGGGGTTTCGTCATG 24660 24661 TTACCCAGGCTGGTCTTGAACTCCTGGGCTCAAGCAATCTGCCCACCTTGGCCTGCCAGA 24720 24721 GTGGTAGAATTACAGGCATGAGTCACTACACCTGGCCGGCAGAGCTAACTTTAGTTAGAA 24780 24781 CAAGGTCATAGGAGAGAGACGTTCAAAGCCTTAACTTGGGGCCAGGTGCAGTGGCTCACA 24840 24841 CCTGTAATCCTAGCACTCTGGGAGGCCAAGGCGGGTGGATTGCCCGAGCTCAGGAGTTTG 24900 24901 AGACTAACCTGGCCAAGACGGTGAAACCCCATCTCTACTGAAAATACAAAAAATTAGCCA 24960 24961 GGCGCGATGGCGCAAACCTGTAGTCCCAGCTACTCAGGAGTCTGTGATGGGAGAATCACC 25020 25021 TGAGCCTGGGGAGATCAAGGATGCATGAACTGTGATTGCACCACTGCACTCCAGCCTGGA 25080
Appendix B Primer design details from PrimerQuest
Appendix C Cutting patterns of HpaII for wild type and mutant HEXA WILD TYPE: MUTANT: