ABCMdb

PMID: 20384480

Rocchi L, Braz C, Cattani S, Ramalho A, Christan S, Edlinger M, Ascenzioni F, Laner A, Kraner S, Amaral M, Schindelhauer D
Escherichia coli-cloned CFTR loci relevant for human artificial chromosome therapy.
Hum Gene Ther. 2010 Sep;21(9):1077-92., [PubMed]

Sentences

No. Mutations Sentence Comment

48 ABCC7 p.Val470Met For construction of the intermediate construct CF1-Met, primers MetF and MetR (Table 1) were used for the V470M exchange by site-directed mutagenesis in a plasmid containing an intron 9 and exon 10 portion during construction of CGT21, and primers E10XcF and CF10NMR (Table 1) for the introduction of the silent XmaI variant and intronic NotI site for PAC ligation. Login to comment 93 ABCC7 p.Val470Met ABCC7 p.Val470Met PFGE separation, Table1.OligodeoxynucleotidesUsedinStudy PrimerSequence(50 -30 )LocationReferenceUsedfor: P77-BGGTCGAGCTTGACATTGTAGGpCYPAC2N(reverse)ThisworkPCR,endSequencing P86pCYPAC2NThisworkEndmapping T7TAATACGACTCACTATAGGGpCYPAC2NSchindelhauerandSchwarz, 2002;Laneretal.,2005 PCR R7CCTCTCCCTATAGTGAGTCGpCYPAC2N (reversecomplement ofT7) ThisworkPCR SP6ATTTAGGTGACACTATAGpCYPAC2NAnandetal.,1991LR-PCREnd sequencing ContYACTCTCGGTAGCCAAGTTGGTTYACleftarmThisworkEndmapping/ sequencingLR-PCR Primer2CCGGAATTCCGGACCGCCGCCG CAAGGAATGG YACrightarmThisworkEndmapping/ sequencingLR-PCR CF1-5RGTTCCAATTCTATAAGATTATCAGCFTR50 regionofCF1ThisworkLR-PCREnd mapping/sequencing 50 CFCCCATGGTGGAATAAAGTACCFTR50 regionof5AThisworkEndmapping/ sequencingLR-PCR CF-37FCGTGTTAGGCTGATTTTGCAGCCFTR50 regionThisworkLR-PCR CF-37RCGACCAGCACATAACAACTCAGCCFTR50 regionThisworkLR-PCR CF-18FGATGTCCTGCAACTGGCAGAGCFTR50 regionThisworkLR-PCR CF-18RCAGAGATCTACATGTGAGGGCCFTR50 regionThisworkLR-PCR CF-1FCTCAGAGAGTTGAAGATGGCGCFTR50 regionThisworkLR-PCR CF-1RGTGAGTGAACTCCAAGGGTGGCFTR50 regionThisworkLR-PCR A1RCGAGAGACCATGCAGAGGTCCFTRexon1Thiswork; Laneretal.,2005 RT-PCR CF3FCTTGGGTTAATCTCCTTGGACFTRintron2Thiswork; Zielenskietal.,1991 LR-PCR CF3RATTCACCAGATTTCGTAGTCCFTRintron3Thiswork; Zielenskietal.,1991 LR-PCR CFc3FGGGATAGAGAGCTGGCTTCCFTRexon3ThisworkRT-PCR Sequencing CF4FTCACATATGGTATGACCCTCCFTRintron3Thiswork; Zielenskietal.,1991 PCR CF4RTTGTACCAGCTCACTACCTACFTRintron4Thiswork; Zielenskietal.,1991 PCR CF7bFAGACCATGCTCAGATCTTCCATCFTRintron6Thiswork; Zielenskietal.,1991 LR-PCR CF7bRGCAAAGTTCATTAGAACTGATCCFTRintron7Thiswork; Zielenskietal.,1991 LR-PCR B3FAATGTAACAGCCTTCTGGGAGCFTRexon8Ramalhoetal.,2002RT-PCR Sequencing 1080 In9FCAAGTAGCAGGTGAAGCAAGTGCCFTRintron9ThisworkPCR Sequencing GFP1-ALGAACTTCAGGGTCAGCTTGCGT21,CFTR/EGFP fusionexon10 Laneretal.,2005RT-PCR Sequencing CF10FGCAGAGTACCTGAAACAGGACFTRintron9/exon10Thiswork;Zielenski etal.,1991 PCR CF10RCATTCACAGTAGCTTACCCACFTRintron10Thiswork;Zielenski etal.,1991 PCRLR-PCR MetFa GACTTCACTTCTAATGATGATTATGGGAGAACTGGCFTRintron10ThisworkPCR,V470M exchange MetRa CCAGTTCTCCCATAATCATCATTAGAAGTGAAGTCCFTRexon10ThisworkPCR,V470M exchange E10XcFa GGATTATGCCCGGGACCATTAAAGAAAATATCATCTTTGGsynthetic/CFTR,exon10ThisworkPCR,XmaIsite CF10NMRa CGGTGGTACGCGTGCGGCCGCCCTAACATTTACAGCAATAAsynthetic/CFTR,exon10ThisworkPCR,NotI/MluIsite C16DGTTGGCATGCTTTGATGACGCTTCCFTRexon10(reverse)Ramalhoetal.,2002PCRSequencing RT-PCR CFi10fusGTGACTGCAATTCTTTGATGCCFTRintron10ThisworkPCRSequencing CF11FCAACTGTGGTTAAAGCAATAGTGTCFTRintron10Thiswork;Zielenski etal.,1991 PCR CF11RGCACAGATTCTGAGTAACCATAATCFTRintron11Thiswork;Zielenski etal.,1991 PCRSequencing CF12FGTGAATCGATGTGGTGACCACFTRintron11Thiswork;Zielenski etal.,1991 PCR CF12RCTGGTTTAGCATGAGGCGGTCFTRintron12Thiswork;Zielenski etal.,1991 PCR CF14cRGTATGTGTTCCATGTAGTCACTGCCFTRexon14ThisworkLR-PCR CF14iFTTCTATGGATCATGAGCAGCCACCFTRintron14ThisworkLR-PCR CF17bFTTCAAAGAATGGCACCAGTGTCFTRintron17aThiswork;Zielenski etal.,1991 PCR CF17bRATAACCTATAGAATGCAGCACFTRintron17bThiswork;Zielenski etal.,1991 PCR CF19cFGCAAAATACACAGAAGGTGGAAATGCCCFTRexon19ThisworkLR-PCR CF21FAATGTTCACAAGGGACTCCACFTRintron20Thiswork;Zielenski etal.,1991 PCR CF21RCAAAAGTACCTGTTGCTCCACFTRintron21Thiswork;Zielenski etal.,1991 PCR CFaFCTAGGGTGATATTAACCAGGGCFTRpoly(A)regionThisworkPCR CFaRGAGGCTTGAAGACATTATGCTAGCFTRpoly(A)regionThisworkPCR aM2RGTTGCAGCGCTATGAGGCTTGAAGACATTATGCTAGsynthetic/CFTR,þ2kb(stop)ThisworkCGT21construction LR-PCR aB5Ra GTGTTGCAGCGCGCTTTCAGCTCTGCCGTCTGCCFTR30 region,syntheticThisworkLR-PCR,BssHIIsite CF6-4FCTCTGTGAAGGAGGTTCTAAGAACCFTRþ9.5kb(stop)ThisworkLR-PCREndsequencing CFþ23FTGTCTACTGAGCAGAGGTAGCCFTRþ23kb(stop)ThisworkLR-PCR CFþ23RGTAGCAGAGTCTAGAATTAGTCCCFTRþ23kb(stop)ThisworkLR-PCR 30 CFCTCTGTGATTCACACTGAAAGCFTRþ51kb(stop)ThisworkEndmapping/sequencing LR-PCR AcFGCACTCTTCCAGCCTTCCb-ActinThiswork;Laner etal.,2005 RT-PCR AcRAGAAAGGGTGTAACGCAACTAAGb-ActinThisworkRT-PCR a Modifiedprimers. Login to comment 158 ABCC7 p.Val470Met Sequencing of the cloned exon 10, determined with primers In9F/P77-B, confirmed the right orientation and the presence of the corrected V470M polymorphism, and that the sequence in all three clones had no PCR-derived mutation. Login to comment