7. Appendices
Appendix 1: NCBI ob gene annotation
(updated
1/11/2005)
Annotated sequence file:LOCUS 7 26351 bp DNA HTG 1-NOV-2005
DEFINITION Homo sapiens chromosome 7 NCBI35 partial sequence 127470282..127496632 reannotated via EnsEMBL ACCESSION chromosome:NCBI35:7:127470282:127496632:1 VERSION chromosome:NCBI35:7:127470282:127496632:1 KEYWORDS .
SOURCE Human
ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Catarrhini;
Hominidae; Homo.
COMMENT This sequence was annotated by the Ensembl system. Please visit The Ensembl web site, http://www.ensembl.org/ for more
information.
COMMENT All feature locations are relative to the first (5') base of the sequence in this file. The sequence presented is always the forward strand of the assembly. Features that lie outside of the sequence contained in this file have clonal location coordinates
in the format: .:..
COMMENT The /gene indicates a unique id for a gene,
/note="transcript_id=..." a unique id for a transcript, /protein_id a unique id for a peptide and note="exon_id=..." a unique id for an
exon. These ids are maintained wherever possible between versions. COMMENT All the exons and transcripts in Ensembl are confirmed by
similarity to either protein or cDNA sequences. FEATURES Location/Qualifiers source 1..26351 /organism="Homo sapiens" /db_xref="taxon:9606" gene 5001..21351 /gene=ENSG00000174697 mRNA join(5001..5029,15714..15885,18127..21351) /gene="ENSG00000174697" /note="transcript_id=ENST00000308868" CDS join(15742..15885,18127..18486) /gene="ENSG00000174697" /protein_id="ENSP00000312652" /note="transcript_id=ENST00000308868" /db_xref="HUGO:LEP" /db_xref="CCDS:CCDS5800.1" /db_xref="Uniprot/SWISSPROT:OB_HUMAN" /db_xref="RefSeq_peptide:NP_000221.1" /db_xref="RefSeq_dna:NM_000230.1" /db_xref="Uniprot/SPTREMBL:Q4TVR7_HUMAN" /db_xref="Uniprot/SPTREMBL:Q6NT58_HUMAN" /db_xref="EntrezGene:3952" /db_xref="AgilentCGH:A_14_P126175" /db_xref="AgilentProbe:A_32_P112263" /db_xref="EMBL:AF008123" /db_xref="EMBL:AY996373" /db_xref="EMBL:BC060830" /db_xref="EMBL:BC069323" /db_xref="EMBL:BC069452" /db_xref="EMBL:BC069527"
/db_xref="EMBL:D49487" /db_xref="EMBL:D63519" /db_xref="EMBL:D63710" /db_xref="EMBL:DQ054472" /db_xref="EMBL:U18915" /db_xref="EMBL:U43415" /db_xref="EMBL:U43653" /db_xref="GO:GO:0005179" /db_xref="GO:GO:0005615" /db_xref="GO:GO:0006112" /db_xref="GO:GO:0007165" /db_xref="GO:GO:0007267" /db_xref="IPI:IPI00012760.1" /db_xref="MIM:164160" /db_xref="MIM:601665" /db_xref="PDB:1AX8" /db_xref="UniGene:Hs.194236" /db_xref="protein_id:AAA60470.1" /db_xref="protein_id:AAB63507.1" /db_xref="protein_id:AAC31660.1" /db_xref="protein_id:AAC50400.1" /db_xref="protein_id:AAH60830.1" /db_xref="protein_id:AAH69323.1" /db_xref="protein_id:AAH69452.1" /db_xref="protein_id:AAH69527.1" /db_xref="protein_id:AAX81413.1" /db_xref="protein_id:AAY46797.1" /db_xref="protein_id:BAA08448.1" /db_xref="protein_id:BAA09787.1" /db_xref="protein_id:BAA09839.1" /db_xref="AFFY_HG_Focus:207092_at" /db_xref="AFFY_HG_U133A:207092_at" /db_xref="AFFY_HG_U133A_2:207092_at" /db_xref="AFFY_HG_U133_PLUS_2:207092_at" /db_xref="AFFY_HG_U95Av2:35895_at" /db_xref="AFFY_HuGeneFL:D49487_s_at" /db_xref="AFFY_HuGeneFL:U43653_at" /db_xref="AFFY_U133_X3P:207092_3p_at" /db_xref="AFFY_U133_X3P:g4557714_3p_at" /translation="MHWGTLCGFLWLWPYLFYVQAVPIQKVQDDTKTLIKTIVTRIND ISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLE NLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQ LDLSPGC" exon 18127..21351 /note="exon_id=ENSE00001208755" exon 5001..5029 /note="exon_id=ENSE00001422382" exon 15714..15885 /note="exon_id=ENSE00001303768" ORIGIN 1 – 4200 continuous sequence
4201 gcctaccaca ccactcacag gctatgatga caaaaacgtg gctacat^ctg ggaccacccc (4248)(c/g)(dbSNP:rs791619)
4261 ccaaccccca ctttgtacgt ag^gaaatacg gagttgagga tggagaccca cagtatgtcc (4283)(g/a)(dbSNP:rs6976701)
4321 agagtgtccc caaaggccac agtgcccgcc tggagccctc cagagagcgt gcactccctg 4381 gggtgccagc cagagacaac ttgccctgag gcttggaact cgattctccg ^cgtgccagag (4431)(c/t)(dbSNP:rs10262600)
4441 aaggggtggg acttcagaac ccccaacccc gcaatctggg tcggggagcc tggcgcactg 4501 cgggccgctc cctctaaccc tgggcttccc tggcgtccag ggccgtcggg gccgagtccc 4561 gattcgctcc caccccgaag ccgcgccagg accaacgagg gcgcagccgt atgccccagc 4621 ccgctccgcg gagcccctca cagccacccc cgccccgacc gcgccccgcg cggctcgaag 4681 caccttccca aggggctggt ccttgcgcca tagtcgcgcc ggagcctctg gagggacatc>
ob promoterF 4741 aaggatttct cgctcctacc agccaccccc aaatttttgg gaggtaccca agggtgcgcg 4801 cgtggctcct gg^cgcgccga ggccctccct cgaggccccg cgaggtgcac actgcgggcc (4813)(c/a)(dbSNP:rs791620)
4861 cagggctagc agccgcccgg cacgtcgcta ccctgag>ggg cggggcggga gctggcgcta
ob ex1F
4921 gaaatgcgcc ggggcctgcg gggcagttgc gcaagt^t<gtg atcgggccgc tataagaggg (4957)(branch site - YRYRAY) ob promoterR
ob-Exon1 (5001-5029 -> 29bp) TOP
4981 gcgggcaggc atggagcccc GTAGGAATCG CAGCGCCA^GC GGTTGCAAGg taaggccccg
(5019)(g/a)(dbSNP:rs2167270)
5041 gcgcgctcct tcctccttct ctgctggtct ttcttggcag gccacagggc cccacacaac 5101 tctggatccc ggggaaactg agtcaggagg gatgcagggc <ggatggctta gttctggact
ob ex1R
5161 atgatagctt tgtaccgagt tctagccaga tagaaggtta ccgggagctg gggagc^gttg (5217)(g/a)(dbSNP:rs2278814)
5221 gatttgctgc tgggctgtgc cggtgcccag aaggcaggac cttgcagaac cagccaggtc 5281 cctgggagac tgtcagaccc accaacctgg tggcattcgc agagctgaga tgcattggaa 5341 attgccttgg gcacatcccc aaagatcagg atgtcccacc ccagtctgaa ggagataaag 5401 ttgggggtag gagagacgca gatgcaagtg atcagtctca gtcccagaca ttgccttgct 5461 ctgcgggtag gaattcagga ttcattttcc agggaagttc ctgacctctg aatgagaggg 5521 – 15180 continuous sequence
15181 ggtgagtagc ataatcgcta atgatagggt aatgatagag cacatttcac aacaccttta 15241 agccctttca cgtgcatcag ataatttgat cctcataaaa gcctagagat agatatatta 15301 cagggatgaa ggtggagtat tttgtggtta tgtgatatgt ttaaaattat gcagtgagta 15361 aatgactggg ttcaaaccag accttaaaag tctgttatct ttccctcgag catgcaatga 15421 agtctacatc atccctacca tgtccatttg atcacaccct ggcctcacag ctctgtggtc 15481 tacaggatac ctcatggtgg ttttattgac cagacaataa tcctctttct aaggggatgc 15541 atttcattaa tacatatgta gatcatgaat tgtctttgac tttgagggga tggtagccag 15601 agcagaaagc aaagctgatt ttcatccccg tctggtaatg tggttggt>aa tgtgaagatg
ob ex2F
ob-Exon2 (15714-15885 -> 172bp) TOP
15661 ggtgtattct gagataccgg ctccttgcag tgtgtggttc cttctgtttt cagGCCCAAG 15721 AAGCCCATCC TGGGAAGGAA A^ATGCATTGG GGAACCCTGT GCGGATTCTT GTGGCTTTGG start
15781 CCCTATCTTT TCTATGTCCA AGCTGTGCCC ATCCA^AAAAG TCCAAGATGA CACCAAAACC (15816)(a/g)(dbSNP:rs13306517)
15841 CTCATCAAGA CAATTGTCAC CAGGATCAAT GACATTTCAC ACAC^Ggtaag gagagtatgc (15885)(g/a)(dbSNP:rs1800583)
15901 ggggacaaag tagaactgca gccagcccag ca^ctgg<ctcc tagtggcact ggacccagat
(15933)(c/t)(dbSNP:rs13306518) ob ex2R
15961 agtccaagaa acatttattg aacgcctcct gaatgccagg cacctactgg aagctgagaa 16021 ggatttgaaa gcacagggct ccactctttc tggttgtttc ttttggcccc tctgcctgct 16081 gagattccag gggttagtgg ttctaattct aaaccactcc aagaacattt gattttgcta 16141 catgtttcca tttaaaaatc ataggatttg ggctgggtgt ggtggcttgt acctgtcatc 16201 – 17820 continuous sequence
17821 aaataaaaca tatgaaacca tggctggttc ttcagcagag gccatgtaga gaaaggaatg 17881 acctaggaaa gttggcctgg aagtggagg^g aaggatggtg tgggaaaagc aggaatctcg (17910)(g/a)(dbSNP:rs6956510)
17941 gagaccagct tagaggcttg gcagtcacct gggt^gcagga tacaagggcc tgagccaaag (17975)(g/a)(dbSNP:rs3828942)
18001 tggtgaggga gggtggaagg aggcagccca gagaatgacc ctccatgcc>c acggggaagg
ob ex3F
18061 cagagggctc tgagag^cgat tcctcccaca tgctgagcac ttgttctccc tcttcctcct
(18077)(c/t)(dbSNP:rs17151914)
ob-Exon3 (18127-21351 -> 3225bp) TOP
18121 gcatagCAGT CAGTCTCCTC CAAACAGAAA GTCACCGGTT TGGACTTCAT TCCTGGGCTC 18181 CACCCCATCC TGACCTTATC CAAGATGGAC CAGACACTGG CAGTCTACCA ACAGATCCTC 18241 ACCAGTATGC CTTCCAGAAA C^GTGATCCAA ATATCCAACG ACCTGGAGAA CCTCCGGGAT (18262)(g/a)(dbSNP:rs17151919)
18301 CTTCTTCAC^G TGCTGGCCTT CTCTAAGAGC TGCCACTTGC CCTGGGCCAG TGGCCTGGAG (18310)(g/a)(dbSNP:rs1800564)
18361 ACCTTGGACA GCCTGGGGGG TGTCCTGGAA GCTTCAGGCT ACTCCACAGA GGTGGTGGCC 18421 CTGAGCAGGC TGCAGGGGTC TCTGCAGGAC ATGCTGTGGC AGCTGGACCT CAGCCCTGGG 18481 TGC^TGAGGCC TTGAAGGTCA CTCTTCCTGC AAGGACTA^CG TTAAGGGAAG GAACTCTGGC stop, (18519-18768)(STS:35281)
18541 TTCCAGGTAT CTCCAGGATT GAAGAGCATT <GCATGGACAC CCCTTATCCA GGACTCTGTC
ob ex3R2
18601 AATTTCCCTG ACTCCTCTAA GCCACTC^TTC CAAAGGCATA AGACCCTAAG CCTCCTTTTG (18628-18823)(STS:78152)
18661 CTTGAAACCA AAGATATATA CACAGGATCC TATTCTCACC AGGAAGGGGG TCCACCCAGC 18721 AAAGAGTGGG CTGCATCTGG GATTCCCACC AAGGTCTTCA GCCATCAACA AGAGTTGTCT 18781 TGTCCCCTCT TGACCCATCT CCCCCTCACT GAATGCCTCA ATGTGACCAG GGGTGATTTC 18841 AGAGAGGGCA GAGGGGTAGG CAGAGCCTTT GGATGACCAG AACAA^GGTTC CCTCTGAGAA (18886)(g/t)(dbSNP:rs17151922)
18901 TTCCAAGGAG TTCCATGAAG ACCA<CATCCA CACACGCAGG AACTCCCAGC AACACAAGCT
ob ex3R
18961 GGAAGCACAT GTTTATTTAT TCTGCATTTT ATTCTGGATG GATTTGAAGC AAAGCACCAG 19021 CTTCTCCAGG CTCTTTGGGG TCAGCCAGGG CCAGGGGTCT CCCTGGAGTG CAGTTTCCAA 19081 TCCCATAGAT GGGTCTGGCT GAGCTGAACC CATTTTGAGT GACTCGAGGG TTGGGTTCAT 19141 CTGAGCAAGA GCTGGCAAAG GTGGCTCTCC AGTTAGTTCT CTCGTAACTG GTTTCATTTC 19201 TACTGTGACT GATGTTACAT CACAGTGTTT GCAATGGTGT TGCCCTGAGT GGATCTCCAA
19260 – 26352 continuous sequence
Appendix 2:
NCBI obR gene sequence annotation (updated 1/11/2005)
Annotated sequence file:LOCUS NC_000001 232191 bp DNA linear CON 25-OCT-2004
DEFINITION Homo sapiens chromosome 1, complete sequence. ACCESSION NC_000001 REGION: 65593007..65825197
VERSION NC_000001.8 GI:51511461 KEYWORDS HTG.
SOURCE Homo sapiens (human) ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Catarrhini;
Hominidae; Homo.
REFERENCE 1 (bases 1 to 232191)
AUTHORS International Human Genome Sequencing Consortium. TITLE Finishing the euchromatic sequence of the human genome JOURNAL Nature 431 (7011), 931-945 (2004)
PUBMED 15496913
COMMENT GENOME ANNOTATION REFSEQ: Features on this sequence have been produced for build 35 version 1 of the NCBI's genome annotation [see documentation].
On Aug 24, 2004 this sequence version replaced gi:42406218. The DNA sequence is part of the third release of the finished
human reference genome. It was assembled from individual clone sequences by the Human Genome Sequencing Consortium in
consultation with NCBI staff. FEATURES Location/Qualifiers source 1..232191 /organism="Homo sapiens" /mol_type="genomic DNA" /db_xref="taxon:9606" /chromosome="1" misc_feature <1..509 /standard_name="RP11-86K19" /note="FISH-mapped clone" gene <1..567 /gene="DNAJC6"
/note="Derived by automated computational analysis using gene prediction method: GNOMON. Supporting evidence includes similarity to: 2 mRNAs, 21 ESTs"
/db_xref="GeneID:9829" /db_xref="MIM:608375" mRNA <1..567
/gene="DNAJC6"
/product="DnaJ (Hsp40) homolog, subfamily C, member 6" /note="Derived by automated computational analysis using gene prediction method: GNOMON. Supporting evidence includes similarity to: 2 mRNAs, 21 ESTs"
/transcript_id="XM_375737.1" /db_xref="GI:42655781" /db_xref="GeneID:9829" /db_xref="MIM:608375" misc_feature <1..18123 /standard_name="RP5-992N21" /note="FISH-mapped clone" misc_feature <1..147615 /standard_name="RP4-630A11" /note="FISH-mapped clone" gene 5334..17279 /gene="OBRGRP"
gene prediction method: BestRefseq. Supporting evidence includes similarity to: 1 mRNA"
/db_xref="GeneID:54741"
mRNA join(5334..5438,10001..10076,14560..14746,16501..17279) /gene="OBRGRP"
/product="leptin receptor gene-related protein"
/note="Derived by automated computational analysis using gene prediction method: BestRefseq. Supporting evidence includes similarity to: 1 mRNA"
/transcript_id="NM_017526.2" /db_xref="GI:41327153"
/db_xref="GeneID:54741"
CDS join(5423..5438,10001..10076,14560..14746,16501..16617) /gene="OBRGRP"
/go_component="integral to plasma membrane"
/go_function="molecular_function unknown [pmid 9207021]" /go_process="biological_process unknown [pmid 9207021]" /codon_start=1
/product="leptin receptor gene-related protein" /protein_id="NP_059996.1"
/db_xref="GI:8923785" /db_xref="GeneID:54741" gene 10001..222191
/gene="LEPR"
/note="Derived by automated computational analysis using gene prediction method: BestRefseq. Supporting evidence includes similarity to: 1 mRNA"
/db_xref="GeneID:3953" /db_xref="MIM:601007" mRNA join(5333..5438,10001..10076,150244..150303,155171..155500, 157024..157147,177355..177563,181146..181291, 183358..183502,186090..186380,186541..186658, 189736..189935,193451..193599,194645..194804, 194912..194994,200706..200922,202662..202844, 204626..204721,206051..206156,207604..207679, 214900..215120,217850..220126,220889..222191) /gene="LEPR" /product="leptin receptor"
/exception="unclassified transcription discrepancy" /note="Derived by automated computational analysis using gene prediction method: BestRefseq. Supporting evidence includes similarity to: 1 mRNA"
/transcript_id="NM_002303.2" /db_xref="GI:40254463" /db_xref="GeneID:3953" /db_xref="MIM:601007" ORIGIN 1 - 4920 continuous sequence
4921 gggggcgggg tcgggaccag cagggacggc ctctgagagt tgggggcgga attctgggcc 4981 ggtgagggct ggggcgggac cagcagggga ggcctccgcg agctagggga ggagctctgc 5041 attgtctggg gcggggcttc gagtagcggt agcgaggtct ggagccgcag gcgctgcctc 5101 cgcgaggtag gggaggagct ctgtactgtc aggggcgggg ctctgagtag cgaggccggg 5161 tctgaactat caggcgcggc ctctgcgagc tgggggcggg gttctgcacc ggcgggggcg 5221 gggttgtgag tggtgggggc ggggtctgga gcagcaggcg ccgcgtttgc gagctaaggt
obR-Exon1(5333-5438 -> 106bp)
5281 cggagttctg caccggcggg ggcggggctc tgcgtggccg gggcgggctc ggGATCCGCG 5341 GGGCGACTCC CGGTCTGGCT TGGGCAGGCT GCCCGGGCCG TGGCAGGAAG CCGGAAGCAG 5401 CCGCGGCCCC AGTTCGGGAG AC^ATGGCGGG CGTTAAAGgt acatcgcggt ccccggctcg Start (of OBGRP)
5521 gcgccgttgg ggaacggcct caccaccctt cccgcctctc cggttcggga ggcgatcgac 5581 cgctcccttc gtcccttggg gtgtgggtgg agcggcgttt cgggggagcc ttggcccttt 5641 – 9720 continuous sequence
9721 aatttgggac ctttttttct ttctttttta ttttagtaaa tgtttttatt gataaatgat 9781 tcacatacca taaaattaac attcttaaag tgtaagattt catggttttt agtatattca 9841 gaaggttatg cagccatcac tactatctaa ttccagaaaa tttactcatc cgccaaagaa 9901 actctggacc tattagaagt cactccccat ttccccaaac cctctagtgc ctgacaacct
obR-Exon2(10001-10076 -> 76bp)
9961 ctactgtggg aa^ctttaact tttggcttta tttttcacag CTCTCGTGGC ATTATCCTTC (9973)(branch site - YNYYRAY)
10021 AGTGGGGCTA TTGGACTGAC TTTTCTTATG CTGGGATGTG CCTTAGAGGA TTATGGgtaa 10081 gttatcattt caaaaagaac tattcctctt tctgtgtctt tgtcactatt agtatgggtg 10141 ttagagagtt cggtcaattt agcaccaagt tctaaccagt gagttagtgg agcccagttt 10201 atgaacacag tccctttgtg ggtcaggtgt acttttgcag agggctctgt taaaaattga
10261 – 14220 continuous sequence
14221 gacactctac tatgttctat agggaaggca gaaatgacca agttaaaatt ctgtctttaa 14281 agaaattgtg gctcagtggt acagaaaaca gtaaagtttc tagggtatat attatatagt 14341 ggcttcttaa cacaatataa gctacctaca tagtaatttt aaaattatca ctacttatct 14401 atccctggta gaaatcatct tatgttctaa tattcacaat taattaattt tttgacctaa 14461 acatttaaaa tagcaatgat tttgaaatag tagtatgtct ttcatttaaa a^tgtaactgt (1451
OBRGRP-Exon3(14560-14746 -> 187bp)
2)(branch site - YRYRAY)
14521 tacttttctt tttggatttt gcctgggtcc aactgacagC GTTTACTGGC CCTTATTCGT 14581 CCTGATTTTC CACGCCATCT CCCCCATCCC CCATTTCATT GCCAAAAGAG TCACCTATGA 14641 CTCAGATGCA ACCAGTAGTG CCTGTCGGGA ACTGGCATAT TTCTTCACTA CTGGAATTGT 14701 TGTTTCTGCC TTTGGATTTC CTGTTATTCT TGCTCGTGTG GCTGTGgtaa gttttatttt 14761 ctattgtttt gcccaaccgt tgctgagttt acttcagagg cctgtgtctg ggacctccat 14821 ttcatgctca ttacttaggc tttatactca aggccgtgtg tttttagttt ctctgttcct 14881 ctactttaga cctgcctaac agtagttcgg ccatgccttc agagtgcttt ctttatttct 14941 cttacaactt ttagccagac gatatgattt cttacttttt ctactttaag ctattctctg
15001 – 16320 continuous sequence
16321 catcacagaa gtaattagac t^cattactga acagttcatt gtctcctgtt acattattaa (16342-16467)(STS:SHGC-32733)
16381 gccttttagc agctttgtgt aacatttcat tggatgtttc taatgcagaa aataataggg 16441 attgcaaaga g^tacaatatg aaggaagtaa ggatttaatc cttcttttct tgtctttcag (16452)(branch site - YRYRAY)
OBRGRP-Exon4(16501-17279 -> 779bp)
16501 ATCAAATGGG GAGCCTGCGG CCTTGTGTTG GCAGGCAATG CAGTCATTTT CCTTACAATT 16561 CAAGGGTTTT TCCTTATATT TGGAAGAGGA GATGATTTTA GCTGGGAGCA GTGG^TAGCAC stop
16621 TTTATTCTGA TTACAGTGCA TTGAATTTCT TAGAACTCAT ACTATCTGTA TACATGTGCA 16681 CATGCGGCAT TTTACTATGA AATTTAATAT GCTGGGTTTT TTAATACCTT TATATATCAT 16741 GTTCACTTTA AGAAAGACTT CATAAGTAGG AGATGAGTTT TATTCTCAGC AAATAGACCT 16801 GTCAAATTTA GATTATGTTA CTCAAATTAT GTTACTTGTT TGGCTGTTCA TGTAGTCACG 16861 GTGCTCTCAG AAAATATATT AACGCAGTCT TGTAGGCAGC TGCCACCTTA TGCAGTGCAT 16921 CGAAACCTTT TGCTTGGGG^A TGTGCTTGGA GAGGCAGATA ACGCTGAAGC AGGCCTCTCA (16940-17077)(STS:RH79027)
16981 TGACCCAGGA AGGCCGGGGT GGATCCCTCT TTGTGTT^GTA GTCCATGCTA TTAAAAGTGT (17018-17192)(STS:G20438;A005R18)
17041 GGCCCACAGA CCAAGAGCCT CAACATTTCC TAGAGCCTTA TTAGAAATGC AGAATCTGAA 17101 GCCCCACTCT GGACCCAGGA CATTTTGATG AGATCCAAAG GAGTTGTATG C^ACATGAAAG (17152-17303)(STS:RH68452)
17161 TTTGAGAAGC ATCATCATAG AGAAGTAAAC ATCACACCCA ACTTCCTTAT CTTTCCAGTG 17221 GCTAAACCAC TTAACCTCTC TGGGTGTTAC CTGCTCATTT GTTTAAAAAA AAAAAAAAAg 17281 tctcacctgc tttcatgctg aggacaagtt cagatgttca agcctataat atttaggcag 17341 ttcctcaaat ttatgaaaag tgttctcaga attgggagac agtcaaaggg tacaaagcct 17401 cagttaggag gaataagtgt gatttttttt taaagatcac ttgcacagca tgctaaatat 17461 aggaataatt gaatgtatat ttcaatattg ctaagagagt aaatttctaa tgttctcata
17521 – 150000 continuous sequence
150001 taaaggaaca ttcagtttaa attttttcga cttgtatttt taatgatgta agataggatg 150061 cgtacaggaa taaatctgta gcctaatttt acgtgagata tttctcagat attgatctag 150121 agtatcacat gtaaatttag agacttatct ataatccctt tccttttatg ttttccacag 150181 acaacttata tatatgtgtt tttgtttttg tgtgtg^ttct gattttagat ttaccttttc (150217)(branch site - YNYYRAY)
obR-Exon3(150244-150303 -> 60bp)
150241 cagGTGTACT TCTCTGAAGT AAG^ATGATTT GTCAAAAATT CTGTGTGGTT TTGTTACATT
start
150301 GGGgtaagtt atttgctcat ttgctgtttt aatgccctta aacatggtag agattttgcc 150361 tttagagatg ctgttttatt tattagctaa cagaatttcc tatttctagt taggaattct 150421 catacatgct tatgattaaa aatgcaaacc atagtatagg tgtatcagtg tgaaaacgaa 150481 aacccttccc aacctcctaa agacaacctg atggtgtatc cttccagaat cctctctctc 150541 – 155040 continuous sequence
155041 agacactcac tgaggactta gaaagttaaa taattattga gcactacatg gtttaatctc 155101 agatactttc tattcatgtc ttagtcaaaa tgattacttt tttctatgtg tctttttaat
obR-Exon4(155171-155500 -> 330bp)
155161 a^tcctaacag AATTTATTTA TGTGATAACT GCGTTTAACT TGTCATATCC AATTACTCCT (155162)(branch site - TCCTRAY(U12 intron))
155221 TGGAGATTTA AGTTGTCTTG CATGCCACCA AATTCAACCT ATGACTACTT CCTTTTGCCT 155281 GCTGGACTCT CAAAGAATAC TTCAAATTCG AATGGACATT ATGAGACAG>C TGTTGAACCT
obR ex4F 155341 AAGTTTAATT CAAGTGGTAC TCACTTTTCT AACTTATCCA AA^ACAACTTT CCACTGTTGC
(155383)(a/g)(dbSNP:17127716)
155401 TTTCGGAGTG AGCAAGATAG AA^ACTGCTCC TTATGTGCAG ACAACATTGA AGGAAAGACA (155423)(a/g)(dbSNP:1137100)
155461 TTTGTTTCAA CAGTAAATTC TTTAGTTTTT <CAACAAATAG gtaagcatta gctat^gtttt
(155516)(g/t)(dbSNP:2186248) obR ex4R
155521 aaatgtattg aacaatgttt tttaattctt tgatacctgt atgaaataga tgtcttatgg 155581 ctatgatttt aacatacata atcattttta tatggataat aaaattagga ttcattatgt 155641 gaacaaacta ataaataatt tccaaattat cttgtatccc ataaaaatat aatgagggaa 155701 ttattttact tttaagtaca ttccttgtag tcttctaaat ttgctttgaa atagaaaaac 155761 – 156780 continuous sequence
156781 aataattgaa tttaattcta ttactttaga aaaaaggaaa ctgagggcca gagggactaa 156841 gtgatgggca aacgcatgcc actagttaaa gcttgttctt tttctctcag aataggcaat 156901 ggaagttgta tcagaacact ggtaaatttg atttagctct tctctttcac tgagttgttc 156961 agatggtttt tgagatttca tgtagttgtt tttttttttt tttttttttt tttttaaatt
obR-Exon5(157024-157147 -> 124bp)
157021 cagATGCAAA CTGGAACATA CAGTGCTGGC TAAAAGGAGA CTTAAAATTA TTCATCTGTT 157081 ATGTGGAGTC ATTATTTAAG AATCTATTCA GGAATTATAA CTATAAGGTC CATCTTTTAT 157141 ATGTTCTgta agtaccaaat aattaatttg gcatttctaa tacacagttt ttttaaaaga 157201 gctttcatat acggaagtag attataaacc tcttgcatcc ctacatttcc tattatatgt 157261 tttatttttt aatatagcat tctgctctgt taggtttcct cttttctcct cctgctaatt 157321 gaggtagcaa gacagtcagg tttagggtca cgattgtcag gtccatgagt ataccaaatg 157381 – 177180 continuous sequence
177181 acctgctgta atctttatct ttgtttttct aatgtagggt tttttttttt cagataccct 177241 ttaagctggg tgtcccaaat agtttacttc aattagtatt tagtatcctg ctttaaaagc
obR-Exon6(177355-177563 -> 209bp)
177301 ctatccagta ttttcatatc tg^ttttaata tttagctctt atttttcaat atagGCCTGA (177323)(branch site - YNYYRAY)
177361 AGTGTTAGAA GATTCACCTC TGGTTCCCCA AAAAGGCAGT TTTCAGATGG TTCACTGCAA 177421 TTGCAGTGTT CATGAATGTT GTGAATGTCT TGTGCCTGTG CCAAC>AGCCA AACTCAACGA
obR ex6F
177481 CACTCTCCTT ATGTGTTTGA AAATCACATC TGGTGGAGTA ATTTTCC^AGT CACCTCTAAT (177528)(a/g)(dbSNP:1137101)
177541 GTCAGTTCAG CCCATAAATA TGGgtaagtt atgcactaaa atgatgataa taggtctaaa 177601 catcagtcat atataaaggt taaaaattgc ttacaaaaat atttgctagc ttatctcact 177661 ttgcttaaca ctgtaatgat ggtagatgta gta<ctggggg tattaagagt ggcttctaga
obR ex6R
177721 atgatttaac aatggtatgt atatctctgc cattgtcact taaattctgt tttgaaaact 177781 gttttctttc aatcctggat ctatgtaatg gatgt^atatt gattggatat cactttttca
(177816)(a/g)(dbSNP:4655537)
177841 catctcagat aactattttt gaaaatag^ta gcatgtttct tgcctgaatt tattccttca (1777869)(''/taga)(dbSNP:10665816)
177901 ataaatattt cttagaggct catgtttgtc agagactgct ccaggagctg gaaaaagagt 177961 – 180900 continuous sequence
180901 ctgattcata gtctttacca cttaagggga gggtaatgaa tatacatgga ttgctatgat 180961 aaggtacaag gacgagaccc agtattcaag cttgtttcgg ttctataatt agtccttgga 181021 taaagtcacc ttttaagtac ttgaaaggca agatatgatg aaaatttatc ttgactttat 181081 tttattcagc ^tataattgtc atgaaaatta cttgacttaa aagtattctc ttttttttcc (181091)(branch site - YRYRAY)
obR-Exon7(181146-181291 -> 146bp)
181141 ttaagTGAAG CCTGATCCAC CATTAGGTTT GCATATGGAA ATCACAGATG ATGGTAATTT 181201 AAAGATTTCT TGGTCCAGCC CACCATTGGT ACCATTTCCA CTTCAATATC AAGTGAAATA 181261 TTCAGAGAAT TCTACAACAG TTATCAGAGA Agtaagtata ttttagtaag taaaaggaaa 181321 agttgagaag taaataaaga ccctcttaag tcccatagca attaccctct gcatactaaa 181381 tatatacatt tcttctaaag cagaatgaat tataattcaa catttcatat tatatatcat 181441 atatagatag atatacgtat gtatatatat ggctatagga aatctagtta cagaaacttg 181501 aattcataat tacaattatt ttccaccaac agaccactga tttcatcttt tttttaattg 181501 – 183120 continuous sequence
183121 gtatttgtta attatcatta tgattttttc ttggacccat gggctattta aaattttatt 183181 gactaatgtg caaacttttt ttttggttat tgatgtttgt tttaattccg ctgtggccag 183241 ataactactg tgtaagatat taattctttg aaatttgatg agattagctt atcctcactt
obR-Exon8(183358-183502 -> 145bp)
183301 ttagtaaagg ttcca^catca acttgatgtt ctgatgtttt aatataatat ttaacagGCT (183316)(branch site - YNYYRAY)
183361 GACAAGATTG TCTCAGCTAC ATCCCTGCTA GTAGACAGTA TACTTCCTGG GTCTTCGTAT 183421 GAGGTTCAGG TGAGGGGCAA GAGACTGGAT GGCCCAGGAA TCTGGAGTGA CTGGAGTACT 183481 CCTCGTGTCT TTACCACACA AGgtaggtta tgtaatagcc acttctactg ggagggaaat 183541 atatttcctg aacttgcctt tgtatagtat aagcatctta cattttgagg ccttaaaatg 183601 aaaggaggaa cacagtatca acttccttgt ttaaactttg aacaggccaa tttgtgaatt 183661 aaaattctgt cagagcctct aggcttgtgt gtttctattg cagtatattg taggatcact 183721 tttcctatta aaaagaaaaa attaatataa cagctccaaa ttaacaaact cagaaagcca 183781 – 185881 continuous sequence
185881 aagctcaact cactttagaa tgagagcact cgggacacta gataactgta cacaaacagg 185941 taatttcagc attatccagt ttttaaatta cactcatttt atttttggca gtgtaactct 186001 ggaatgtgtt gtgaatattt ttcgatgtgg tacaaattac ttacagaatg ttttgtcttc
obR-Exon9(186090-186380 -> 291bp)
186061 at^ctgatatc ctttcttccc tcattacagA TG>TCATATAC TTTCCACCTA AAATTCTGAC
(186063)(branch site - CTRAY) obR ex9F
186121 AAG^TGTTGGG TCTAATGTTT CTTTTCACTG CATCTATAAG AAGGAAAACA AGATTGTTCC (186124)(c/t)(dbSNP:3790419)
186181 CTCAAAAGAG ATTGTTTGGT GGATGAATTT AGCTGAGAAA ATTCCTCAAA GCCAGTATGA 186241 TGT<TGTGAGT GATCATGTTA GCAAAGTTAC TTTTTTCAAT CTGAATGAAA CCAAACCTCG
obR ex9R
186301 AGGAAAGTTT ACCTATGATG CAGTGTACTG CTGCAATGAA CATGAATGCC ATCATCGCTA 186361 TGCTGAATTA TATGTGATTG gtaagaaaac agaggtttt^g ttcattttgt ^tccacaactt (186400)(a/g)(dbSNP:13306522), (186411)(c/t)(dbSNP:17097193)
186421 gaattttcat tatggaccct ccttatatta gagtcctgtt aaagatgtaa gaaaaaattt 186481 ttttcattga attttttgga gtttttgctt ta^tattaata ttttaatatg tttcaaatag (186513)(branch site - YNYYRAY)
obR-Exon10(186541-186658 -> 118bp)
186541 ATGTCAATAT CAATATCTCA TGTGAAACTG ATGGGTACTT AACTAAAATG ACTTGCAGAT 186601 GGTCAACCAG TACAATCCAG TCACTTGCGG AAAGCACTTT GCAATTGAGG TATCATAGgt 186661 acgtattatt tttgctgttt tgtttttctg tgggcacagt gagataaaaa ttttctttag
186721 aaatattaca acagtagtct tacagattat ttgcttcctg aaagaggaaa gtataatata 186781 atgaatcagg aaatttttga gggattatat aaattggtga ttcttgaaaa atattcacac
186841 – 186840 continuous sequence
189541 agtggaacag tttcatcccg aaatcctgaa ccatctccca tcccccatta gtggaaaaat 189601 tgtcttccat gaaacc^ggtc cctggtgcca aaaaggttgg ggactgctgt tttaaacaac
(189617)(a/g)(dbSNP:11208686)
189661 aaatcagaat tctcagatat cttcttgttg cttt^tattaa tgtatactaa ttgactattt
(189695)(branch site - YNYYRAY) obR ex11F
obR-Exon11(189736-189935 -> 200bp)
189721 ttgtatct>tt taaagGAGCA GCCTTTACTG TTCTGATATT CCATCTATTC ATCCCATATC 189781 TGAGCCCAAA GATTGCTATT TGCAGAG<TGA TGGTTTTTAT GAATGCATTT TCCAGCCA^AT
<TGC AGGTTTTTAT GAATGCATTT TCCAGCC
(189838)(a/g)(dbSNP:13306526) obR ex11R
189841 CTTCCTATTA TCTGGCTACA CAATGTGGAT TAGGATCAAT CACTCTCTAG GTTCACTTGA 189901 CTCTCCACCA ACATGTGTCC TTCCTGATTC TGTGGgtatg tcaagctgcg ttgtgtcttc 189961 atttgtgtta gcagatttgt atgcagattg atgcagattt aatgttatat tttgccattt 190021 tctgatcaca ttagattttg aaaaaaaaat tgttcctgtt tacagtggta ttgagaaaat 190081 – 193200 continuous sequence
193201 tactagcttt cagagtaaca tgaataatgt attgactttt ttcagtcaag tctatatttg 193261 cgaaaaagag ataaatccct cttttaatat atcacataaa gtaataggga aacaaatgag 193321 ataatgagtg agaaagttat gaagaagata ttgcttgatg aatacagatg tatgaaaata 193381 taacacaatg tttttaggca ttattactat ttaaattacc atcacttaat actattgtaa
obR-Exon12(193451-193599 -> 149bp)
193441 aaatttctag TGAAGCCACT GCCTCCATCC AGTGTGAAAG CAGAAATTAC TATAAACATT 193501 GGATTATTGA AAATATCTTG GGAAAAGCCA GTCTTTCCAG AGAATAACCT TCAATTCCAG 193561 ATTCGCTATG GTTTAAGTGG AAAAGAAGTA CAATGGAAGg taccttttac ttagaacttc 193621 agctttcctc attaaatgct atttttataa tatgtaagag tttaattgca ttagattaat 193681 tttattggca taaaagtaca ttctcctgta ttgtgtagct attttcatta aatagattta 193741 – 194400 continuous sequence
194401 caagtaaacc agttcaaaat cttgtcatca tcagcaaggg agtgacaaga taaataagaa 194461 aatccttgac ttgaaggagt ttacaatcca agcctaattg tgactatttc tgaaggcaga 194521 gaacacagaa tcagttctct taattttgga atagtgttaa ggtttaaaat aaaatgtact 194581 tcagggccct ttagatacat atgtgttggt aatgatcaat ctaatg^cttt gacttatttt (194627)(branch site - YNYYRAY)
obR-Exon13(194645-194804 -> 160bp)
194641 acagATGTAT GAGGTTTATG ATGCAAAATC AAAATCTGTC AGTCTCCCAG TTCCAGACTT 194701 GTGTGCAGTC TATGCTGTTC AGGTGCGCTG TAAGAGGCTA GATGGACTGG GATATTGGAG 194761 TAATTGGAGC AATCCAGCCT ACACAGTTGT CATGGATATA AAAGgtctgc agagattttg 194821 taaatgtgtt ttgaaagtgc ataagtgtgt gcttcaaata tggctgaaaa gtatttcttc
obR-Exon14(194912-194994 -> 83bp)
194881 aaaaacatat a^cacaacttg tcattttgca g>TTCCTATGA GAGGACCTGA ATTTTGGAGA
(194892)(branch site - YRYRAY) obR ex14F
194941 ATAATTAATG GAGATACTAT GAAAAA^GGAG AAAAATGTCA CTTTACTTTG GAAGgtattc (194967)(c/g)(dbSNP:8179183)
195001 ccaattttaa tattaatctt aaattgtatt tttatactct taaaaattta cttcatggtc 195061 cataatcctg ttattaatct tc<ggaaagct caacaatcct gtattttcat tgcatttatg
obR ex14R
195121 agatgtattt aaagagagct aagatgtaca gtggctgaag cactggcata gtgttaggag 195181 actttcattc tcttcttagc ttcacatatt atagttacgt cagtactatt tgggcaaagt
195241 – 200520 continuous sequence
200521 atggagactg tggcagaggc aaactctatc ccacccatct ccccagcctc tcagccttgt 200581 aatagtacct gccctgatct ggcccctgcc tcctaataag aaatgtagaa tgagagttat 200641 gtgagctcag ttagtataaa aagcactgca gcccttaaa^c taatcaattt ctatatttac (200680)(branch site - CTRAY)
obR-Exon15(200706-200922 -> 217bp)
200761 ATACTTCCTG CAATGGAACA TGGTCAGAAG ATGTGGGAAA TCACACGAAA TTCACTTTCC 200821 TGTGGACAGA GCAAGCACAT ACTGTTACGG TTCTGGCCAT CAATTCAATT GGTGCTTCTG 200881 TTGCAAATTT TAATTTAACC TTTTCATGGC CTATGAGCAA AGgtaagaag aggtacagag 200941 tggtaatcca ttgcctcttt taatatttaa cttttgcaaa ctctcctatt ttaaattatc 201001 tttcaagcag cctgcaattc tctgcatctg aaagtctctc tttcaccttt catttccttt 201061 gtctggaaaa ataacaaagt acattgtaca atgatcacat aatacagtcc ttgagctttc 201121 – 202500 continuous sequence
202501 ctctcatgat gcaaaagtaa ttgtcatgta tcatatttat ttgtgtaaat tgtctgtctt 202561 ctcttcctta ttccctttag taggttataa gttcctcaag agtattagta gatttt^tatt (202617)(branch site - YNYYRAY)
obR-Exon16(202662-202844 -> 183bp)
202621 aatttttaat ttaaaaatta atttccttct tttcccctca gTAAATATCG TGCAGTCACT 202681 CAGTGCTTAT CCTTTAAACA GCAGTTGTGT GATTGTTTCC TGGATACTAT CACCCAGTGA 202741 TTACAAGCTA ATGTATTTTA TTATTGAGTG GAAAAATCTT AATGAAGATG GTGAAATAAA 202801 ATGGCTTAGA ATCTCTTCAT CTGTTAAGAA GTATTATATC CATGgtaagt ttactatact 202861 ttagtaagtt gctctcatgg attaatatga cactacagat tattaattct attaatatag 202921 ccagttaatg aaaacttcaa aaatatagag gatactacca tcctaataca tataatccta 202981 taacctttat caaaacattt aattctcttc ctcttctctt ctcttctctt ttcttttctt 203041 – 204421 continuous sequence
204421 tatttggaaa actattgtca tgactagata attagaagtg cttaagtttg ttttgaaact 204481 cccttgataa tttaatccac aaataataag taatttctat gtttggaaat atatgatagt 204541 cactttaatt cttttttaaa atgtatgttc cactcattac ^tattaatttt gtataaatga (204581)(branch site - YNYYRAY)
obR-Exon17(204626-204721 -> 96bp)
204601 gccttttacg tatttttctc ctcagATCAT TTTATCCCCA TTGAGAAGTA CCAGTTCAGT 204661 CTTTACCCAA TATTTATGGA AGGAGTGGGA AAACCAAAGA TAATTAATAG TTTCACTCAA 204721 Ggtaaaaatt ataattttag tttcctttta caccaatgtg tgtgcctaat ttgtttgcag 204781 taatattgcc atctgattat tctttttctg atagaatcat ggaagagtcc aaagtcttct 204841 – 205860 continuous sequence
205861 tgaacaaaat aataagtatt ttattaatgt gatcacaaca tatttttatc ttcatttttt 205921 atttttatat ttttgaaggt aataagagat ttgtgatgaa ttcagaaaat gtctactata 205981 atttttgata cagaaagaaa ttaatatttc ctcaagtttc tgagttgtgt aaattgtatt
obR-Exon18(206051-206156 -> 106bp) obR ex18F
206041 tctttttcag ATGATATTGA AAAACACCAG AGTGATGCAG GTTTATATGT AATTGTGCCA 206101 ^GTAATTATTT CCTCTTCCAT CTTATTGCTT GGAACATTAT TAATATCACA CCAAAGgtat
(206101)(c/g)(dbSNP:6693573)
206161 tgtacttgag gttaagaatc tttacggcaa aa<gtccttac gcgtattcaa accctgattt obR ex18R
206221 aaaaccttct aaattaggaa tattaaagtc ttctaagtgc ttttatatgt agtctgtata 206281 caattaagat agcataaaaa ggaaaggcct gttgt^gagga aatggcattc cattttgcta
(206316)(c/g)(dbSNP:4655743)
206341 ggactgtaag agttaaagaa tgaggaaaga aacacgaaac acagcttaac agttaaagac
206401 – 207480 continuous sequence
207481 tctgacccta cggaggcata gttgatctgg tggactaatt tctagcttgt ctgttcacta 207541 actgttcaca t^tttcaatat ggatgttttt ctctaatttt gatgccctgt ttatcctttg (207552)(branch site - YNYYRAY)
obR-Exon19(207604-207679 -> 76bp)
207601 tagAATGAAA AAGCTATTTT GGGAAGATGT TCCGAACCCC AAGAATTGTT CCTGGGCACA 207661 AGGACTTAAT TTTCAGAAGg ttgctttttc aatttttttt aacccagaat atatacgatt 207721 gcaatctaga cgccatatga cttatagagc attaagcatt tttaagattt aaaaggtcat 207781 attttattca attcatctta atatatctgt tattatatac tttcaagaat agcaggatgc 207841 agggatttga aatcaggaag aaacaaattt cagcgcatag tttatcgtat gagagttcac 207901 – 214680 continuous sequence
214681 atgcgataag tctccatctc tccatgcctg aactgccttt tattctgccc tcctcgattg 214741 ttagtttggt ggagtatcaa ttcttagaaa tagcctttcc aagagaacat tgagataatt
214801 cctgccttaa catatagtat ccagtgttgc ttctgagcag tcagatacta t^tgtgatttt (214852)(branch site - YRYRAY)
obR-Exon20 transcript variant 3(214900-215120 -> 221bp)
214861 tactcctcac tgtttactta taatttcctt tttaaacagA TGCTTGAAGG CAGCATGTTC 214921 GTTAAGAGTC ATCACCACTC CCTAATCTCA AGTACCCAGG GACACAAACA CTGCGGAAGG 214981 CCACAGGGTC CTCTGCATAG GAAAACCAGA GACCTTTGTT CACTTGTTTA TCTGCTGACC 215041 CTCCCTCCAC TATTGTCCTA TGACCCTGCC AAATCCCCCT CTGTGAGAAA CACCCAAGAA 215101 ^TGATCAATAA AAAAAAAAAA gaaagaaaaa aaaattctcc tcatttttgt tgttttgaat (215103-215106)(STS:tv3-Stop)
215161 tttcagtgat gaatctggag tggttctttt tttatttaat gtggttattt tttgaagaga 215221 ctttcaattc catgtctcct gtcaattcta agaaattata ttatttcttt gataatttcc 215281 tctcctcttg tttctttgtt tctctttcta gaatctgtat gttggatgat ggaccctctg 215341 – 217620 continuous sequence
217621 attaaagctg tccttagagt gtgatattaa aacattcatt aagaaaatgg gagaagccac 217681 cttagttcaa aattttgccc tttcccaaaa ggatgcatta ttgtaaccta acacaaaaat 217741 ttatagtcca gaacccatgc ttgacaatgt tttttgaaat cttttatctt tttctttgtg
obR-Exon20 transcript variant 2(217850-220126 -> 2277bp)
217801 agtgattttt tattttgctt tcttattttg ttttatttta tctaaacagA GAACGGACAT 217861 TCTT^TGAAGT CTAATCATGA TCACTACAGA TGAACCCAAT GTGCCAACTT CCCAACAGTC (217867-217870)(STS:tv2-Stop)
217921 TATAGAGTAT TAGAAGATTT TTACATTTTG AAGAAGGGGA GCAAATCTAA AAAAAATTCA 217981 GTTGAACTTC TGAGAGTTAA CATATGGTGG ATTATGTTGA TTTAGAACTT AAAATAGATG 218041 TGTAAATTTG GGTTCAAAAT GTAGATTTGA GTCCAGTTTG GATGTGTGAT TAATTTTCAA 218101 – 220740 continuous sequence
220741 tatatgtatt ttagtcaaaa actagttaat ttgtggttga cttatgttct ttattattaa 220801 cttaacacac ttccatttct gccagtatga cataatgaag caaaatt^ttt taacataatt (220848)(branch site - YNYYRAY)
obR-Exon20 transcript variant 1(220889-222191 -> 1303bp)
220861 gagccttaaa atgtgtcttt tcttttagCC AGAAACGTTT GAGCATCTTT TTATCAAGCA 220921 TACAGCATCA GTGACATGTG GTCCTCTTCT TTTGGAGCCT GAAACAATTT CAGAAGATAT 220981 CAGTGTTGAT ACAT^CATGGA AAAATAAAGA TGAGATGAT^G CCAACAACTG TGGTCTCTCT (220995-221497)(STS:LEPR-2), (221020-221257)(STS:PMC209419P1)
221041 ACTTTCAACA ACAGATCTTG AAAAGGGTTC> TGTTTGTATT AGTGACCAGT TCAACAGTGT obR ex20F
221101 TAACTTCTCT GAGGCTGAGG GTACTGAGGT AACCTATGAG GACGAAAGCC AGAGACAACC 221161 CTTTGTTAAA TA^CGCCACGC TGATCAGCAA CTCTAAACCA AGTGAAACTG GTGAAGAACA
(221173)(c/t)(dbSNP:1805095)
221221 AGGGCTTATA AATAGTTC^AG TCACCAAGTG CTTCTCTAGC AAAAATTCTC C^G<TTGAAGGA (216239)(a/g)(dbSNP:6413506),(216272)(a/g)(dbSNP:1805096) <TTGACGGA
obR ex20R
221281 TTCTTTC^TCT AATAGCTCAT GGGAGATAGA GGCCCAGGCA TTTTTTATAT TATCAGATCA TTCTTTC TCT AATAGC
(221288-221652)(STS:PMC310709P1)
221341 GCATCCCAAC ATAATTTCAC CACACCTCAC ATTCTCAGAA GGATTGGATG AACTTTTGAA 221401 ATTGGAGGGA AATTTCCCTG AAGAAAATAA TGATAAAAAG TCTATCTATT ATTTAGGGGT 221461 CACCTCAATC AAAAAGAGAG AGAGTGGTGT GCTTTTGACT GACAAGTCAA GGGTATCGTG 221521 CCCATTCCCA GCCCCCTGTT TATTCACGGA CATCAGAGTT CTCCAGGACA GTTGCTCACA 221581 CTTTGTAGAA AATAATATCA ACTTAGGAAC TTCTAGTAAG AAGACTTTTG CATCTTACAT 221641 GCCTCAATTC CAAACTTGTT CTACTCAGAC TCATAAGATC ATGGAAAACA AGATGTGTGA 221701 CCTAACTGTG ^TAATTTCACT GAAGAAACCT TCAGATTTGT GTTATAATGG GTAATATAAA tv1 stop
221761 GTGTAATAGA TTATAGTTGT GGGTGGGAGA GAGAAAAGAA ACCAGAGTCA AATTTGAAAA 221821 – 222670 continuous sequence
Appendix 7:
DNA extraction method (protocol on p. 101)
Extraction Buffers and chemicals used:
Nuclear Lysis Buffer (1l, pH 8.2 set with 1M NaOH, store at 4
°C)
1.211g
Tris-Cl
0.01M
23.4g
NaCl
0.4M
0.6g
EDTA
0.002M
Cell Lysis Buffer (1l, pH 7.4 set with concentrated HCl, store at 4
°C)
8.3g
NH
4Cl
0.155M
1.1g
KHCO
30.01M
0.03g
EDTA
0.0001M
Phosphate Buffered Saline (PBS) (1l, store at 4
°C)
2g
KCl
0.027M
8g
NaCl
0.137M
1.14g
Na
2HPO
40.008M
0.2g
KH
2PO
40.0015M
10% SDS (100ml, store at room temperature to prevent precipitation)
10g
SDS
Work in vapour hood
Proteinase K (store at -20
°C)
10mg/ml dissolved in dH
2O, aliquot in 1.5mL Eppendorf tubes
6M NaCl (500ml, saturated solution)
Genomic DNA Extractions from Blood:
• Place ~10ml blood in a 50ml Falcon tube
• Add 30ml cold Lysis Buffer
• Place on ice for 15 min, shaking each 5 min interval
• Centrifuge @ 1500rpm for 10min
• Pour off supernatant , keeping pellet
• Add 10ml PBS and mix
• Centrifuge @ 1500rpm for 10min
• Pour off supernatant keeping pellet
• Dissolve pellet in :
30ml Cell – Lysis buffer
30μl Proteinase K (10mg/ml)
300μl 10% SDS
• Mix well and incubate overnight in 55°C waterbath
__________________________________________________________
Day 2:
• Add 1ml 6M NaCl and shake for 1min
• Centrifuge @ 3500rpm for 30 min
• Transfer supernatant to new tube and shake for 15s
• Centrifuge @ 2500rpm for 15 min
• Transfer supernatant to new tube without foam or pellet
• Add 2 volumes ice cold 100% ethanol to precipitate DNA
• Scoop out DNA and place in Eppendorf tube with 500μl 70% ethanol
• Centrifuge @ 14 000rpm for 10 min @ 4°C
Appendix 8:
Rapid DNA extraction protocol
Rapid DNA Isolation from 300μl Whole Blood:
*PureGene
®DNA Isolation Kit (Gentra Systems™, Minneapolis, USA)
Rapid 25 Minute Protocol, does not include RNase treatment
Cell Lysis:
1. Add 300μl whole blood to 900μl RBC Lysis Solution and incubate for 1min at room
temperature; invert gently 10 times during incubation.
2. Centrifuge for 20s at 13 000 –16 000g, Remove supernatant (leave approximately 10μl
liquid) and vortex to resuspend the pellet.
3. Add 300μl Cell Lysis Solution and pipette up and down.
Protein Precipitation:
4. Add 100μl Protein Precipitation Solution and Vortex at high speed for 20s
Centrifuge for 1 min at 13 000 –16 000g (dark brown pellet forms)
DNA Precipitation:
5. Transfer supernatant to a clean Eppendorf tube containing 300μl 100% isopropanol and
mix by inverting samples 50 times.
6. Centrifuge at 13 000 – 16 000g for 60s (get small white pellet)
7. Pour off supernatant and drain tube on clean absorbent paper.
Add 300μl 70% ethanol and invert tube several times to wash pellet.
8. Centrifuge at 13 000- 16 000g for 60s
Carefully pour off ethanol and invert and drain tube for 30 min on clean towel paper.
DNA Hydration:
Appendix 9:
Multiphor SSCP/HD protocol (description on p. 104)
Multiphor Solutions:
4
0% acrylamide PDA solution: 1l(keep in foil in fridge) 396g acrylamide
4g Piperazine diacrylamide bring to volume (1l) with dH2O
0,75M TRIS- Formate Buffer: 1l
90.8g TRIS
600ml dH2O (dissolve)
pH = 9.0 with formic acid bring to volume (1l) with dH2O
41% Glycerol: 100mL
41ml glycerol 59ml dH2O
TRIS- Borate Buffer: 1l (pH = 9.0)
125.9g TRIS 17.3g Boric acid 700ml dH2O (dissolve)
50μl Bromophenol Blue (4% solution) bring to volume (1l) with dH2O
SSCP loading Dye: 50ml
47.5ml 95% Formamide 0.16g 100mM NaOH
0.125g 0.25% Bromophenol Blue 0.125g 0.25% Xylene cyanol bring to volume (50ml) with dH2O
10% APS: 1.5mL
0.15g Ammonium Persulphate bring to volume (1.5ml) with dH2O
Gel mix: X10 (12% non-denaturing)
53ml 40% acrylamide-PDA 85ml Tris-Formate Buffer 30ml 41% Glycerol keep in foil in fridge
Per 1 Gel: 15ml Gel Mix 150μl 10% APS 15μl TEMED Plate Glue 50μl 0.25% plate glue
(3-(Trimethoxysilyl) propyl methacrylate diluted in 100% ethanol
Silver Staining solutions:
Solution I: 1l0.1% silver nitrate = 0.3g silver nitrate 500ml dH2O
keeps max 2 days in the dark
Solution II : 1l
4.5g NaOH 1L dH2O
Gel Plates preparation (do everything in quick succession) • Wash plates (118mm x 220mm x 3mm) 4X with 70% ethanol
• Every 2 runs clean plate with blocks with 80μl Repelcote (Merck, Darmstadt, Germany) especially over blocks
• Put 80μl plateglue and 6μl 10% acetic acid on plate without blocks • Wipe well until resistance is felt and wash a few times with ethanol • Clean spacers and place them on plates
• Camp well on both short sides and long area close to blocks and then place upside down on bench • Prepare gel mix and pour quickly using a syringe
• Allow to set at room temp for 30min
• Separate plates (gel should be on one without blocks) • Put water on surface of multiphor apparatus
• Don’t get water on plates when placing on multiphor apparatus • Slide plate facing upwards over water (no bubbles)
• Use 3 buffer strips/gel-side/2h run soaked in TRIS-borate buffer • Place strips on both sides of gel close to the wells
• Wash electrodes of multiphor gently with 70% ethanol each time before use
PCR Products:
• Take required volume of PCR product and add 3μl SSCP loading dye (all on ice) • Digest @ 95°C for 5min in PCR thermocycler
• Place on ice immediately for 3min
• Load 3μl (skipping ends of gel) onto gel and run @ 12°C first ( if resolution not good, run at 9°C) • 2-2.5h at 355V
Visualization of bands:
• remove gel from apparatus
• rinse X2 in dH2O ( remove buffer because it interferes with the stain)
• incubate for 10min @room temp in solution I (silver nitrate intercalates with DNA) • rinse X2 with dH2O
• incubate for 10min @room temp in solution II • rinse X2 with dH2O
• blot dry with paper towel • cover with filter paper