Anemia

Research Article

Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

Table 4

Primers and conditions for PCR on genomic DNA of the coding sequence plus intron/exon boundaries of FANCA, FANCC, FANCE, FANCF, FANCG, and FANCB.
(a)
FANCA
Primer nameSequence (5′ > 3′)Product length (bp)
FANCA_ex1Fgtaaaacgacggccag GCGCCTCCCCCAGGACCAACA362
FANCA_ex1Rcaggaaacagctatga AGGCTCTGGCGGGAAGGGATCGG
FANCA_ex2Fgtaaaacgacggccag CTCTTCGGGAGGGTGTCGCTGGT328
FANCA_ex2Rcaggaaacagctatga CTCTTCGGGAGGGTGTCGCTGGT
FANCA_ex3Fgtaaaacgacggccag GCCTGGCCTGGAGCTTGAAT392
FANCA_ex3Rcaggaaacagctatga CGCAGGTTGAATCAGACGCTGTT
FANCA_ex4Fgtaaaacgacggccag TAAGGCATTTTAAACAGCAAGTC430
FANCA_ex4Rcaggaaacagctatga TGCCAATAAATACTGAGCAAACT
FANCA_ex5Fgtaaaacgacggccag AGTATTGTTTCAGGTAATTTGTT356
FANCA_ex5Rcaggaaacagctatga TGAAGGTACTTCTTTCCAATCCA
FANCA_ex6Fgtaaaacgacggccag AGATGTGTTTCAGGCTCTAAGTT402
FANCA_ex6Rcaggaaacagctatga GCAATGCAATCTAGTCTAGTACA
FANCA_ex7Fgtaaaacgacggccag TGGGATTTAGTTGAGCCTTACGTCTGC421
FANCA_ex7RcaggaaacagctatgaAAGGTGAATGGAAACACTTAAACTCATGTCA
FANCA_ex8Fgtaaaacgacggccag GTGGTCAGGTGAGCAGTAACTTC401
FANCA_ex8Rcaggaaacagctatga TAAATAGGTACAAACAGCACGTT
FANCA_ex9Fgtaaaacgacggccag TTCTCTTGTGTGATGCAGGTATC332
FANCA_ex9Rcaggaaacagctatga TGACCCACAGATTCATGAGGTAT
FANCA_ex10Fgtaaaacgacggccag TTTTGATTAAGGCCTACAGATTG406
FANCA_ex10Rcaggaaacagctatga CCTCCTCCTCACGCACGTTATCG
FANCA_ex11Fgtaaaacgacggccag TTTCAAGTCTGTGGTTATAGTGG410
FANCA_ex11Rcaggaaacagctatga AGACGTAAAAGAGGTCCTAGAAT
FANCA_ex12Fgtaaaacgacggccag CTGTGGGGCTGGTCCTTAACAAA236
FANCA_ex12Rcaggaaacagctatga AGGCAGCATGACAAGTACTAGGC
FANCA_ex13Fgtaaaacgacggccag ACATTGGTTTGCTTGGATATTGA377
FANCA_ex13Rcaggaaacagctatga CTGACAAAGAATGTTCCATCGAC
FANCA_ex14Fgtaaaacgacggccag TGCTGTAATTGCTGTGTAGTCTT411
FANCA_ex14Rcaggaaacagctatga ACTCACATGACAGAGAATCAGGT
FANCA_ex15Fgtaaaacgacggccag ACTACAGCAGCCGCCCGGACACT430
FANCA_ex15Rcaggaaacagctatga GCAGATCTGCAGGAGGCTCTTGG
FANCA_ex16Fgtaaaacgacggccag TCCCAGGCAGTTCCCAGACTAAC312
FANCA_ex16Rcaggaaacagctatga AGCTGATGACAAATCCTCGTAGA
FANCA_ex17Fgtaaaacgacggccag ACCGCTCCCTCCTCACAGACTAC334
FANCA_ex17Rcaggaaacagctatga AAGGCTGAAAAACTCAACTCAAG
FANCA_ex18Fgtaaaacgacggccag GCGCACAGCATGTGGGCCTTTAC397
FANCA_ex18Rcaggaaacagctatga GCAGCTGCTAGAGGCCTTTTCGG
FANCA_ex19Fgtaaaacgacggccag GTGCACAAGAAGACTTCATAATG284
FANCA_ex19Rcaggaaacagctatga AGTCCTTGCTTTCTACACAACTG
FANCA_ex20Fgtaaaacgacggccag CTTCTCTGTGTTGCAGCATATTC298
FANCA_ex20Rcaggaaacagctatga AGAAGAAACCTGGAAGTAGTCAT
FANCA_ex21Fgtaaaacgacggccag ATAATAGATTTGGGGATTGTAAT255
FANCA_ex21Rcaggaaacagctatga CAACAGACACTCAAGGTTAGGAA
FANCA_ex22Fgtaaaacgacggccag TGCAGTGAAGAGTCCTGTTGAGT305
FANCA_ex22Rcaggaaacagctatga ACACACCAGCCTGATGTCACTAT
FANCA_ex23Fgtaaaacgacggccag CAGTCAGCAGGATCCGTGGAATC416
FANCA_ex23Rcaggaaacagctatga GGCCCTGGAACATCTGATACGAC
FANCA_ex24Fgtaaaacgacggccag CCTTCCTGCTGCTCCCGTCC229
FANCA_ex24Rcaggaaacagctatga CAGACTTGGCCCAGCAAGAG
FANCA_ex25Fgtaaaacgacggccag CCGCTGGTGGTTGGATTAGCTGT296
FANCA_ex25Rcaggaaacagctatga TTTCCAGGGCACTGAAGACGAAT
FANCA_ex26Fgtaaaacgacggccag AGCTTGGAAGAGGGCAGTCTGCT347
FANCA_ex26Rcaggaaacagctatga CTCTTCTAATTTTATCAAACGAG
FANCA_ex27Fgtaaaacgacggccag AGACTGTCTCACAACAAACGAAC356
FANCA_ex27Rcaggaaacagctatga CGGTCCGAAAGCTGCGTAAAC
FANCA_ex28Fgtaaaacgacggccag GTTGATGGTCTGTTTCCACCTGA401
FANCA_ex28Rcaggaaacagctatga GAAGGAACGGTCACCTACGTGCT
FANCA_ex29Fgtaaaacgacggccag GACATGGAGGACTGCGTATGAGA411
FANCA_ex29Rcaggaaacagctatga GTGGCTGTGATGACTGGAACGTG
FANCA_ex30Fgtaaaacgacggccag CCCGAGCCGCCAGTCTCAACCCA411
FANCA_ex30Rcaggaaacagctatga AAAGGCAGACCCACCCTAAGCTA
FANCA_ex31Fgtaaaacgacggccag GATAAGCCTCCTTGGTCATGGTA406
FANCA_ex31Rcaggaaacagctatga TGGCAATAAATATCTTAATAGCA
FANCA_ex32Fgtaaaacgacggccag TTCCTGTGCCAGCATACTGCTCT359
FANCA_ex32Rcaggaaacagctatga GGGTGGGGACACACAGACAAGTA
FANCA_ex33Fgtaaaacgacggccag TGGGTTTCAGGGTGGTGGTTGCT356
FANCA_ex33Rcaggaaacagctatga GAACCCTTTCCTCAGTAATTCAC
FANCA_ex34Fgtaaaacgacggccag CGCCCAGGGAAGCCGTTAAGTTT333
FANCA_ex34Rcaggaaacagctatga GCGTTCTGAGAAGGCCACGAGAG
FANCA_ex35Fgtaaaacgacggccag TTCCTTCACTCTACTAGTTGTGG311
FANCA_ex35Rcaggaaacagctatga TGAGATGGTAACACCCGTGATGG
FANCA_ex36Fgtaaaacgacggccag CCATCTCAGCCACCCTCATCTGT350
FANCA_ex36Rcaggaaacagctatga AGGCGCCCACCACCACGAGAACT
FANCA_ex37Fgtaaaacgacggccag GACTTGGTTTCTATGGCGTGGTT310
FANCA_ex37Rcaggaaacagctatga CCCAGAGAAATAGCACTGATTGA
FANCA_ex38Fgtaaaacgacggccag GTTTTCTAAGATCCACTTAAAGG362
FANCA_ex38Rcaggaaacagctatga CTCACTCACACTTCCGCAAACAC
FANCA_ex39Fgtaaaacgacggccag CTGTCCAGAGGCCCAGTATTACC387
FANCA_ex39Rcaggaaacagctatga AGGAGGGCTCGTTCTTAACCATT
FANCA_ex40Fgtaaaacgacggccag GGTGTCCCCAGCACTGATAATAG353
FANCA_ex40Rcaggaaacagctatga AGACATAGTGACAAATGGCTACA
FANCA_ex41Fgtaaaacgacggccag CCCTTGGCATCACCTGCTACCTT403
FANCA_ex41Rcaggaaacagctatga AACAGGCAAACTCACAGGTTAGA
FANCA_ex42Fgtaaaacgacggccag ACCAGCCCTGTTTCTGTATGTCT248
FANCA_ex42Rcaggaaacagctatga ACATGGCCCAGGCAGCTGTCAAT
FANCA_ex43Fgtaaaacgacggccag TGTGGGGGACATGAGAATTGACA378
FANCA_ex43Rcaggaaacagctatga GTAATCCACTTTTTAGTGCAACA
FANCAIVS10Fgtaaaacgacggccag TTTACATGTGCATCAGTTAGCTT184
FANCAIVS10Rcaggaaacagctatga CATGAAGACACAGAAAAAGTAGGT
(b)
FANCC
Primer nameSequence (5′ > 3′)Product length (bp)
FANCC_ex1F
FANCC_ex1R		gtaaaacgacggccag ACCATTTCCTTCAGTGCTGGACA
caggaaacagctatga CCATCGGCACTTCAGTCAATACC		378
FANCC_ex2F
FANCC_ex2R		gtaaaacgacggccag CTAAACAAGAAGCATTCACGTTC
caggaaacagctatga GGAGAAAGGTTCATAATGTAAGC		303
FANCC_ex3F
FANCC_ex3R		gtaaaacgacggccag TCAGCAGAAAGAGAATGTGCAAA
caggaaacagctatga AACATCATAGAACTGGATTCCAC		405
FANCC_ex4F
FANCC_ex4R		gtaaaacgacggccag TGTACATAAAAGGCACTTGCATT
caggaaacagctatga TCCCATCTCACATTTCTTCCGTA		380
FANCC_ex5F
FANCC_ex5R		gtaaaacgacggccag AGAACTGATGTAATCCTGTTTGC
caggaaacagctatga TTACTGCTCTGTGAGAGTTGAGA		367
FANCC_ex6F
FANCC_ex6R		gtaaaacgacggccag GTCTTTGACCTTTTTAGCATGAA
caggaaacagctatga AACGTTTGGACACTGCTGTCGTA		387
FANCC_ex7F
FANCC_ex7R		gtaaaacgacggccag ATTAGTGATTGCATTTTGAACTT
caggaaacagctatga CAAAAATAAAATGTAAATACACG		422
FANCC_ex8F
FANCC_ex8R		gtaaaacgacggccag CTCCTTTGGCTGATAATAGCAAG
caggaaacagctatga CTGATTTTTGAGTTTTTACCTCT		336
FANCC_ex9F
FANCC_ex9R		gtaaaacgacggccag ATACTGCTGAAGCTTATGGCACA
caggaaacagctatga TAACCTTTGTTGGGGCACTCATT		400
FANCC_ex10F
FANCC_ex10R		gtaaaacgacggccag TATGAGGTTATTGGGAGCTTATT
caggaaacagctatga CTGTCTCCCTCATGCTGTAGATA		382
FANCC_ex11F
FANCC_ex11R		gtaaaacgacggccag GAACCAGAAGTAAAGGGCGTCTC
caggaaacagctatga CTGACCTGCTCCAAGCCATCCGT		416
FANCC_ex12F
FANCC_ex12R		gtaaaacgacggccag AAGTACAATTTAAGCCAACCGTT
caggaaacagctatga AGGTTGCCATGACATATGCCATC		451
FANCC_ex13F
FANCC_ex13R		gtaaaacgacggccag CCTCTCTCAGGGGCCAGTGCTTA
caggaaacagctatga AGACCCTCGGACAGGTAACCCAC		435
FANCC_ex14F
FANCC_ex14R		gtaaaacgacggccag ACTTGCTATGCTAATCACCTTGC
caggaaacagctatga AATGCGTGGCCACAGGTCATCAC		437
(c)
FANCE
Primer nameSequence (5′ > 3′)Product length (bp)
FANCE_ex1F
FANCE_ex1R		gtaaaacgacggccag CGCCTCCCTCCTTCCCTTTC
caggaaacagctatga CCCGCCTCCCATACCTGCTAA		540
FANCE_ex2aF
FANCE_ex2aR		gtaaaacgacggccag GCTCTGCCCAGTCTGCCTTGTGC
caggaaacagctatga CTCTGAGTCCTTTCTGCGTTTCC		469
FANCE_ex2bF
FANCE_ex2bR		gtaaaacgacggccag GCCAGAGACAGCTCCAAAGTCTA
caggaaacagctatga CAGCCTTCCCCATGGATAAAGCC		479
FANCE_ex3F
FANCE_ex3R		gtaaaacgacggccag GCCTCTTGACTTTCTTGAATCAT
caggaaacagctatga ACTGTCCTCAGACCTTTACTCCA		352
FANCE_ex4F
FANCE_ex4R		gtaaaacgacggccag TTGAACCAAGTGTAGACTTACCA
caggaaacagctatga GGGAAGGAACCAAGGGCTAAAAG		436
FANCE_ex5F
FANCE_ex5R		gtaaaacgacggccag GTATCTTTTAGCCCTTGGTTCCT
caggaaacagctatga GAATCCCCTCTCTCAAGTACCAC		431
FANCE_ex6F
FANCE_ex6R		gtaaaacgacggccag TTTCCTTTGTAACATGTATCATC
caggaaacagctatga AGCAGAAAGCAGGGAGGCGGTAA		433
FANCE_ex7F
FANCE_ex7R		gtaaaacgacggccag ACAGGCTGGGCATTCTGTTACCG
caggaaacagctatga AGTGAGACACAAGGATCCCCTAA		425
FANCE_ex8F
FANCE_ex8R		gtaaaacgacggccag TTGGAGCAGCAGATAGATACTCA
caggaaacagctatga AGAGGTGGAGCTGAAGTGACCAT		380
FANCE_ex9F
FANCE_ex9R		gtaaaacgacggccag GTTACCTGCCCAGGGTCACCTAG
caggaaacagctatga CTGGCCAGCACTCAGGGTTTTAT		388
FANCE_ex10F
FANCE_ex10R		gtaaaacgacggccag TGGCCTCCTCTCTCCTCAATAGA
caggaaacagctatga AACAGGGAGGCAGTTGCAATCTG		369
(d)
FANCF
Primer nameSequence (5′ > 3′)Product length (bp)
FANCF_ex1aF
FANCF_ex1aR		gtaaaacgacggccag TTTCGCGGATGTTCCAATCAGTA
caggaaacagctatga CTGCACCAGGTGGTAACGAGCTG		449
FANCF_ex1bF
FANCF_ex1bR		gtaaaacgacggccag AGTGGAGGCAAGAGGGCGGCTTT
caggaaacagctatga GCTATCACCTTCAGGAAGTTGTT		456
FANCF_ex1cF
FANCF_ex1cR		gtaaaacgacggccag CCCAAATCTCCAGGAGGACTCTC
caggaaacagctatga TTTCTGAAGGTCATAGTGCAAAC		444
FANCF_ex1dF
FANCF_ex1dR		gtaaaacgacggccag GCTTTTGACTTTAGTGACTAGCC
caggaaacagctatga ATTTGGTGAGAACATTGTAATTT		456
(e)
FANCG
Primer nameSequence (5′ > 3′)Product length (bp)
FANCG_ex1F
FANCG_ex1R		gtaaaacgacggccag AGCCTGGGCGGGTGGATTGGGAC
caggaaacagctatga TCATTTCTGGCTCTTTGGTCAAG		389
FANCG_ex2F
FANCG_ex2R		gtaaaacgacggccag CAGGCCAAGGTAACACGGTTGCT
caggaaacagctatga CCAGTCTCCTCTGTGCCTTAAAC		460
FANCG_ex3F
FANCG_ex3R		gtaaaacgacggccag TATTGTAGCTGTTTTGGTTGGAG
caggaaacagctatga GGTGACAGATGTTGTTTATCCTC		362
FANCG_ex4F
FANCG_ex4R		gtaaaacgacggccag GGAGATGGAGGATGAGGTGCTAC
caggaaacagctatga CGACCACCAACCCAGCCGCCTGT		411
FANCG_ex5F
FANCG_ex5R		gtaaaacgacggccag AGATGGAGATAGGAGAAGACGAG
caggaaacagctatga GCTTCATGAAGGCTGCTTAGTGC		454
FANCG_ex6F
FANCG_ex6R		gtaaaacgacggccag CAGTTCCATGGGCTTCTTAGACC
caggaaacagctatga TCAGGGCTGCAACCAAGTACAAC		393
FANCG_ex7F
FANCG_ex7R		gtaaaacgacggccag GCACTGGGGTCCTGTCACCGTAA
caggaaacagctatga ATAATCTTTGGGAGCCATACTTC		418
FANCG_ex8F
FANCG_ex8R		gtaaaacgacggccag GCTTGTGATGGGGTGACTTGACT
caggaaacagctatga AGTTCAGGTCTAGAAGCAAGGTA		438
FANCG_ex9F
FANCG_ex9R		gtaaaacgacggccag CCTCCTCAGGGCCCATGAACATC
caggaaacagctatga GCAGTGTCTTGAAAGGCATGAGC		400
FANCG_ex10F
FANCG_ex10R		gtaaaacgacggccag CAGGACTCTGCATGGTACCAG
caggaaacagctatga CCAATCAGAAAATCATCCCTC		460
FANCG_ex11F
FANCG_ex11R		gtaaaacgacggccag AGCTCCATGTTCACCTACTTACC
caggaaacagctatga CAGTGCCGCATCTGACTTACATC		397
FANCG_ex12F
FANCG_ex12R		gtaaaacgacggccag AGGATTTGGGGTTTTGGTGACTG
caggaaacagctatga AACTCTTGGGAGCCCTGCATACA		445
FANCG_ex13F
FANCG_ex13R		gtaaaacgacggccag CCGCTTCCATATGTGAGTGTAGG
caggaaacagctatgaC CACAATAGGTCCAAGGACTCTA		340
FANCG_ex14F
FANCG_ex14R		gtaaaacgacggccag CCAAACTAAGGGGTCACATGAAG
caggaaacagctatga GATGGTGAAGCAGAAAGCCCTCC		405
(f)
FANCB
Primer nameSequence (5′ > 3′)Product length (bp)
FANCB_ex3AF
FANCB_ex3AR		gtaaaacgacggccag GATATGGTTATTTGAATTCTTAGCAcaggaaacagctatga GCCATCCTTCATCTCATAGCCTAGT721
FANCB_ex3BF
FANCB_ex3BR		gtaaaacgacggccag ATTAACCTCCCTTACATTGTGATAGcaggaaacagctatga CAATAAGACTCCAGAATGAACTCTA811
FANCB_ex4F
FANCB_ex4R		gtaaaacgacggccag TTTACAAATGACAACTACATGAcaggaaacagctatga TTAAGTATAAAACCACCAATAT391
FANCB_ex5F
FANCB_ex5R		gtaaaacgacggccag ACTGCATCTGGCCTATAGTTcaggaaacagctatga AATACCATTTTTACCCAAGC411
FANCB_ex6F
FANCB_ex6R		gtaaaacgacggccag GTATTTCCTGAATTATTGGTATGTC
caggaaacagctatga CATAAAAGTCCACCATTATAACCTC		395
FANCB_ex7F
FANCB_ex7R		gtaaaacgacggccag TGTTTGGGCCATAAGCCCTA
caggaaacagctatga TTCTGGAGCATCAAGACAGT		355
FANCB_ex8F
FANCB_ex8R		gtaaaacgacggccag GTTGTTTGTATGACATTTAATCATC
caggaaacagctatga ATCATTAAACTCTGCCCATTATCAG		636
FANCB_ex9F
FANCB_ex9R		gtaaaacgacggccag AGGTAATTTTGTTGGCACTT
caggaaacagctatga ATGCGTTCATTCATGCTAGG		531
FANCB_ex10F
FANCB_ex10R		gtaaaacgacggccag AATTGGTTCTGTTTATCATTATGGT
caggaaacagctatga CTACTACAGTAAGCCTCGGTGTTTA		686
PCR conditions:
PCR was performed in Applied Biosystems PE9700 system using 96-well plates. PCR reactions (final volume 25  l) contained 0.5 units Platinum Taq polymerase (Invitrogen), 1,5 mM MgCl2, 0.2 mM NTPs (Invitrogen), and 10 pmol primer.
For the large majority of amplicons, standard PCR conditions were used: preheat 95°C, 5 min, denaturation 95°C, 30 sec, annealing 60°C, 30 sec., elongation 72°C, 1 min, number of cycles: 33.
Fragments with a different annealing temperature were FANCA exons 5, 7, 13, 21, 26, 31, and 38, FANCC exon 7, FANCF fragment 1d and FANCE exon 1 : 55°C; FANCA exon 1 : 64°C. For FANCE exon 1 the PCR mix was supplemented with 10% DMSO.
For FANCB different PCR conditions were used: preheat 95°C, 5 min, denaturation 95°C, 1 min, annealing 50°C, 1 min., elongation 72°C, 1 min., number of cycles 30. For FANCB exon 7 and 9 the annealing temperature was 55°C. For sequencing of exon 7 forward, a special sequencing primer was used: 5′-TTTTTAGAAGGAATGTCTTG-3′.
FA gene specific part of the primer is indicated in capitals. Primers are extended with M13 sequence (indicated in normal letter type), which is used for the sequencing reaction.