LONG PCR AMPLIFICATION OF THE FVIII GENE INTRON 22 GENE INVERSION
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Single tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in hemophilia A. Liu et al (letter) Blood 92, 1458-9, 1998.
Note that this reference contains a mistake in the primer B sequence (correct sequence given later in this protocol). For the correct primer sequences and some other useful tips if you suffer from over-amplification of some of the bands refer to:
Subcycling-PCR for multiplex long distance amplification of regions with high and low GC content: application to the inversion hotspot in the FVIII gene. Liu and Sommer Biotechniques 25, 1022-8, 1998.
Method Validation:
Several samples previously typed by Southern Blotting have been retested by the PCR method, giving concordant results. Six samples have been tested as part of a blinded NEQAS pilot scheme, giving the expected results.
This protocol was submitted by Steve Keeney, Haematology Department, Manchester Royal Infirmary. Any further enquiries should be addressed to:
skeeney@labmed.cmht.nwest.nhs.uk
REAGENTS
Oligonucleotide primers
1. INT22 P 5’-GCCCTGCCTGTCCATTACACTGATGACATTATGCTGAC-3’ (38 mer)
Make a 4 µM stock
2. INT22 Q 5'-GGCCCTACAACCATTCTGCCTTTCACTTTCAGTGCAATA-3' (39 mer)
Make a 4 µM stock
3. INT22 A 5’-CACAAGGGGGAAGAGTGTGAGGGTGTGGGATAAGAA-3’
(36 mer)
Make a 2 µM stock
4. INT22 B 5’-CCCCAAACTATAACCAGCACCTTGAACTTCCCCTCTCATA-3’
(40 mer)
Make a 2 µM stock
note: This primer B sequence differs from that published in the original letter which is incorrect.
Store stocks at -70oC. Working stocks can be made by combining equal volumes of individual primer. If desired, these working stocks can be stored at -20oC.
