[Detection of sickle cell gene by analysis of amplified DNA sequences].
This paper describes a technique of DNA amplification in vitro and its application on detection of sickle cell (Hb S) gene. Genomic DNA was microextracted from dried blood specimen of the first patient with sickle cell trait in China. Target DNA sequence was amplified by the polymerase chain reaction (PCR) with the primers beta 1 (5'-ACACAACTGTGTTCACTAGC-3') and beta 2 (5'-CAACTTCATCCACGTTCACC-3') that primed amplification of an 110-base-pair (bp) segment of beta globin gene. The amplified DNA was digested with a restriction endonuclease Mst II, which has a recognition site at codon 6 in the normal beta globin gene, and cleaved the normal amplified beta globin DNA into two fragments of 54bp and 56 bp which was as an overlap band in agarose gel electrophoresis, while the 110bp fragment amplified from DNA of sickle cell mutation remained uncleaved owing to a single base substitution (A----T) at codon 6 in the mutation. DNA amplification method is rapid, sensitive and simple, and does not require radioactive probes. Besides, the PCR amplification can be carried out on the DNA extracted from dried blood samples. So the technique is very useful for gene diagnosis and carrier screening of genetic disease.
Huang
S Z
SZ
Sheng
M
M
Zhao
J Q
JQ
Qiu
X K
XK
Zeng
Y T
YT
Wang
Q S
QS
He
M X
MX
Zhu
J M
JM
Liu
W P
WP
Li
W W
WW
D000368:Aged; D000755:Anemia, Sickle Cell / Q000235:genetics*; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004252:DNA Mutational Analysis; D005784:Gene Amplification; D006451:Hemoglobin, Sickle / Q000235:genetics*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction
D006451:Hemoglobin, Sickle; D004247:DNA