Most traditional sequencing, or Sanger Dideoxy sequencing
, is performed on PCR products.
These tests have used a range of polymerase chain reaction (PCR)-based methodologies, such as Sanger dideoxy sequencing
, pyrosequencing, and allele-specific real-time PCR.
The draft genome sequence was confirmed by selecting additional primers to generate [approximately equal to] 1 kb products across the entire sequence for direct dideoxy sequencing
(Genewiz, South Plainfield, NJ, USA), applying TaKaRa LA Taq polymerase with GC buffer (TaKaRa Bio, Otsu, Japan) to obtain products in areas that proved difficult to amplify because of potential secondary structures or elevated GC content.
Sanger Sequencing, also called Sanger Dideoxy Sequencing
, is the controlled interruption of enzymatic DNA replication through the use of dideoxynucleotides in primer-directed DNA extension to produce discrete DNA fragments (11,41) (Fig.
After bisulfite treatment, the MS-FLAG target region was PCR-amplified using primers external to the MS-FLAG region, and the amplified products were processed via dideoxy sequencing
The bi-directional dideoxy sequencing
of 20 receptor kinase domains and their adjacent regions revealed novel somatic mutations in fibroblast growth receptor 1 (FGFR1) and frameshift mutations in growth factor receptor alpha (PDFGRA).
Partial 16S rRNA sequences of [approximately equal to] 500 bp of the 5' end, including the V1, V2, and V3 variable regions (13), were determined for other isolates with the same primers, after which direct dideoxy sequencing was performed.
Briefly, partial gene sequences for 6 conserved housekeeping loci (aspC, clpX, fadD, icdA, lysP, and mdh) were obtained by PCR and direct sequenced by automated dideoxy sequencing.
A total of 132 patient DNA samples were genotyped by both the Tag-It assay and DNA dideoxy sequencing
for each of the following six SNPs: factor V Leiden G1691A, factor II G20210A (prothrombin), MTHFR C677T, MTHFR A1298C, factor XIII va1341eu, and TFPI C536T.
These amplification products were cloned into the pGEM-Teasy vector (Promega, Madison, WI) and subjected to automated dideoxy sequencing
(ABI Prism Model 377, Foster City, CA).