4 Vol No4 4 009 8 Life Science Research ug 009 *,, 08 :,,, SYBR Green I,,,, β Ivs--654 (C T) CD7 ( T) : ; ; SYBR Green I; :Q9 : :007-7847(009)04-08-05 Point Mutation Detection Using Ligase-mediated Induced Fluorescence Resonance Energy Transfer MENG Xiang-xian HUNG Jian-hua TN Yong-jun * Institute of Biological Technology State Key Laboratory of Chemo / Biosensing and Chemometrics Hunan University Changsha 08 Hunan China bstract n approach has been developed to genotype point mutations using ligase-mediated induced fluorescence resonance energy transfer FRET When two ligated probes are complementary to template ligase covalently joins the leak of two probes to form a long duplex Duplex inseting dye insects into duplex strands to form induced FRET When there is a mismatch among probes and template FRET can not be induced Using this method the homozygotes and heterozygotes were scored accurately and conveniently This method has been validated with the genotyping of two common point mutations Ivs--654 C T and CD7 T and of β-globin gene in thalassemia disease Key words ligase induced fluorescence resonance energy transfer SYBR Green I thalassemia Life Science Research 009 4 8~87 [5] [6] [8] [9] [~] DSH [] DSH [4] DSH Howell [] 999 [5~] DN : 009-05-9 :009-07-6 : 0505007 008SK085 : 97- * 967- E-mail ytan@yahoocom Tel 07-888
84 009 [] Jobs [] DSH- [] N N 69 β- Ivs--654 [] N N N 5 6- -X- ROX N4 N 5 N5 N6 CD7 SYBR Green I T4 DN ligase 0 T4 DN ligase buffer Taq TakaRa Ex tag 0 ExTaq buffer dntp TP MgCl mresco Solon OH β- Ivs--654 CD7 LS-55 Perkin Elmer mersham Name Template N Template N Probe Ivs--654 N Probe Ivs--654 N4 Probe CD7 N5 Probe CD7 N6 Table Oligonucleotides Sequence 5 5 tctaaagaataacagtgataatttctgggttaaggtaatagcaatatttctgcatataaatatttctgc 5 tctaaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgc ROX-GTTGCTTT p-ccttccc ROX-CTTCCCT p-gcccccg U T4 DN ligase 0 mol / L Tris-HCl ph 76 0 mmol / L MgCl 0 μl 95 5 min 0 5 s 7 % 0 % ImageMaster VDS-CL mersh- 0 mmol / L DTT 0 mmol / L TP mmol / L am spermidine SYBR Green I 50 μl 7 LS-55 00 nmol / L N/N PCR Perkin Elmer 7 00 nmol / L 5 nmol / L 5 nm T4 DN [8] 5 min 0 min 85 5 min 4 s 49 nm
4 : 85 SYBR Green I ROX 5 nm CCTTCTCC- PCR 49 nm 500 nm 700 nm 4 RDB DN PCR - -0 [5] [4] Reverse Dot Blot RDB β-globin Ivs--654 C T CD7 T DN 5 PCR PCR PCR 50 μl 05 U TakaRa Ex Taq Ex Taq Buffer mmol / L MgCl 0 mmol / L dntp Mixture 50 ng DN 0 nmol / L 0 nmol / L Ivs--654 5 -TCTG TCGTG- 5 - GCGTT TTTTGC- CD7 5 -GTCTGCC GTTCTGCCCTG- 5 - TCCCC 94 5 min 94 0 s 50 for Ivs--654 57 for CD7 5 s 70 0 s % SYBR Green I SYBR Green I ROX SGI,, SYBR Green I, Fig Schematic diagram of the assay When the two ligated probes are complementary to template ligase covalently joins the leak of two probes to form a long duplex SYBR Green I dye insects into duplex strands to form induced FRET 50 nm N [] SYBR N N Ivs--654 Green I ROX N N 5 nm 50% 50 nm SYBR Green I N 5 nm ROX 50 nm 5 min ~ N N/N ROX N 5 nm ROX
86 009 0 00 0 50 5 0 500 550 0 650 700 wavelength / nm -654 CD7 4 4 Ivs--654 5 nm 4 SYBR Green I ROX : N+ ; : N/N+ ; : N+ Fig Fluorescence spectra of point mutation detection using ligase-mediated induced FRET Curve N+probes Curve N / N+probes Curve N+probes 4B CD7 5 nm 4 ~ ~ 0 N Marker 0 DN 0 DN 0 DN DN 0 0: N+ ( ); : N+ ; : N / N+ ; : N+ 4 Fig Denaturing polyacrylamide gel electrophoresis Ivs- 0 N+probes without ligase N+probes N / N+ probes N+probes 70 50 0 0 0 t 0 00 00 00 0 t / s 4 β Ivs--654 () CD7 (B) ~ Fig4 Detection of Ivs--654 CD7 B point mutations Curve is mutant homozygote curve is heterozygote curve is wild-type homozygote 70 50 0 0 0 B t 0 00 00 00 0 t / s
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