Direct Sequencing of DNA Fragments Amplified with Single Primers by Polymerase Chain Reaction without Cloning

1996 ◽  
Vol 241 (1) ◽  
pp. 136-139 ◽  
Author(s):  
Masayoshi Iizuka ◽  
Yuki Sugiyama ◽  
Shigeru Iida ◽  
Takao Sekiya
Keyword(s):  
Polymerase Chain Reaction ◽  
Direct Sequencing ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Polymerase Chain

Direct sequencing analysis of transmembrane region of human neu gene by polymerase chain reaction

2006 ◽  
Vol 3 (4) ◽  
pp. 198-201 ◽  
Author(s):  
Hideyuki Saya ◽  
Seiji Ara ◽  
Polly S. Y. Lee ◽  
Jungsil Ro ◽  
Mien-Chie Hung
Keyword(s):  
Polymerase Chain Reaction ◽  
Direct Sequencing ◽  
Sequencing Analysis ◽  
Transmembrane Region ◽  
Chain Reaction ◽  
Polymerase Chain

Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction

2020 ◽  
Vol 58 (4) ◽  
pp. 527-532 ◽  
Author(s):  
Jee-Soo Lee ◽  
Miyoung Kim ◽  
Moon-Woo Seong ◽  
Han-Sung Kim ◽  
Young Kyung Lee ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Circulating Tumor Dna ◽  
Analytical Sensitivity ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Specimen Type ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Ctdna Analysis ◽  
Tumor Dna

AbstractBackgroundChoosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences.MethodsWe isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR.ResultsThe amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts.ConclusionsOur results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

The Investigation of JAK2 and C-MPL Mutations of Myeloproliferative Disorders Patiens

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5261-5261
Author(s):  
Jun Xia ◽  
Yunfeng Shen ◽  
Jianyong Li
Keyword(s):  
Polymerase Chain Reaction ◽  
Direct Sequencing ◽  
Myeloproliferative Disorders ◽  
Jak2v617f Mutation ◽  
Chinese Patients ◽  
Chain Reaction ◽  
Cell Counts ◽  
Allele Specific ◽  
Polymerase Chain ◽  
White Blood Cell Counts

Abstract Most studies have shown that JAK2V617F was found in nearly all the patients with polycythemia vera (PV) and half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). However, JAK2 exon 12 mutations in JAK2V61F-negetive PV patiens and MPLW515L/K mutations in IMF patiens implied other pathogenesis in these myeloproliferative disorders (MPD). Two parts were included in our research to investigate the JAK2 and c-MPL mutations of MPD patients. Firstly, to evaluate the JAK2 exon 12 mutations and its clinical significance in patients with PV, polymerase chain reaction was used to amply the region of JAK2 exon 12 and direct sequencing was performed to detect mutations of JAK2 exon 12. Clinical features of the PV patients with JAK2 exon 12 mutations and JAK2V617F mutation were retrospectively reviewed. The results showed that the JAK2 exon 12 mutations were detected in 3 of 12 JAK2V617F-negtive PV patients (25.0%). Three JAK2 exon 12 mutations were H538QK539L, K539L and N542-E543del respectively. The patients carrying the mutations of JAK2 exon 12 displayed higher hemoglobin(223.6±15.0 vs190.3±20.2g/l, P&lt;0.05), lower white blood cell counts (7.8±2.3 vs16.2±6.7 *109/l, P&lt;0.05)compared with JAK2V617F-positive PV patients (n=31). Secondly, allele specific polymerase chain reaction (AS-PCR) and gene sequencing were used to detected JAK2V617F, MPLW515L and four types of JAK2 exon 12 mutations (F537-K539delinsL, H538QK539L, K539L, N542-E543del) in 30 IMF patients. Results showed that 15 of 30 (50.0%) patients had JAK2V617F mutation and 2 of 30 (6.7%) patients had MPLW515L mutation. JAK2 exon 12 mutations were not detected in IMF patients. Literature reviews show that this is the first report of JAK2 and c-MPL mutations in a number of Chinese patients with BCR/ABL-negative MPD. It is concluded that the JAK2 exon 12 mutations exist in PV patient and the patients with JAK2 exon 12 mutations are characterized by isolated erythrocytosis. The frequency of MPLW515L mutations is high in IMF, which supports recent studies.

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