Mutational analysis by a combined application of the multiple restriction fragment-single strand conformation polymorphism and the direct linear amplification DNA sequencing protocols.
We describe here an improved procedure for polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) for rapid mutational detection. To circumvent the restriction of having to analyze relatively short PCR fragments, restriction endonucleases were used to cleave a longer PCR product and the mixture of fragments was analyzed directly in SSCP gel electrophoresis. This multiple restriction fragment (MRF)-SSCP protocol was demonstrated by the detection of a 4-bp deletion in codons 41-42 and a point mutation in the IVS-2 sequence of the human beta-globin gene. The MRF-SSCP or the standard SSCP protocol was then combined with the linear amplification DNA sequencing (LADS) procedure for direct analysis of the PCR products without further purification for an exact characterization of the mutations detected. In the LADS analysis, homo- or heterozygosity of a mutation was easily distinguished by the appearance of a single- or double-lane band in the sequencing gel. The choice of isotope used and different labeling methods were compared and were found, in some cases, to produce SSCP patterns of different complexities. The combined MRF-SSCP/LADS protocol permits rapid mutational analysis of a large number of clinical samples using only very small amounts of materials and can easily be adopted for nonisotopic clinical applications.
Lee
H H
HH
Department of Medical Research, Veterans General Hospital, Taipei, Taiwan, Republic of China.
Lo
W J
WJ
Choo
K B
KB
D001483:Base Sequence; D004247:DNA / Q000032:analysis; D004252:DNA Mutational Analysis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
D010446:Peptide Fragments; D004247:DNA