The possibility of a valuable resource of circulating DNA for single nucleotide polymorphisms genotyping: the application of a rapid and simple polymerase chain reaction with melting curve analysis for methyltetrahydrofolate reductase polymorphisms.
Circulating DNA from plasma is easily stored and is a valuable resource to access genetic information indispensable to modern hematology. The aim of the present project was to evaluate the integrity of circulating DNA and to investigate whether such DNA is practically tolerable for genotyping single nucleotide polymorphisms (SNP) of methyltetrahydrofolate reductase (MTHFR). We first established a protocol combined with polymerase chain reaction (PCR) and melting curve analysis (MCA) based on the different melting temperatures of heteroduplex amplicons. This method was simple and rapid, requiring 3 hours without any complex manipulation, and allowed for a reliable test and diagnostic validity. The median of the circulating DNA density in 240 donors was 33.5 ng/mL. The DNA consisted of fragments with approxiately 100 to 500 base pairs. Such DNA fragments were acceptable for quantifying the housekeeping genes of - globin using a real-time PCR method and also for genotyping the MTHFR SNP using the method of PCR with MCA. Circulating DNA from storage plasma is acceptable for genetic tests, but it is necessary to note the integrity of DNA.
Mori
Sayaka
S
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences and Central Diagnostic Laboratory, Nagasaki University Hospital, Nagasaki, Japan.
Sugahara
Kazuyuki
K
Uemura
Akiko
A
Yamada
Yasuaki
Y
Uzihara
Kyouko
K
Hayashida
Hideko
H
Ideguchi
Tsuyoshi
T
Ishibashi
Kiyomi
K
Nakazato
Mio
M
Maeda
Takahiro
T
Takamura
Noboru
N
Kamihira
Shimeru
S
D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000097:blood* / Q000235:genetics*; D009691:Nucleic Acid Denaturation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D042965:Methylenetetrahydrofolate Reductase (NADPH2)