ABSTRACT
AIM:To establish a kind of simple and efficient method for cell-free fetal DNA ( cff-DNA) enrich-ment and to investigate its range of applications and the advantages and disadvantages.METHODS:(1) The single nucleo-tide polymorphisms( SNPs) , which linked to paternalβ-thalassemia mutations, were screened.We analyzed the contact be-tween the SNPs inβ-thalassemia gene ( HBB gene) and haploid type by the Haploview software, and then selected these close SNPs which have higher heterozygosity with the HBB gene.(2) We selected 4 cases of different β-thalassemia muta-tions with their husband, and then we used TT-FAST-COLD-PCR to enrich the IVS-II-654 mutations in maternal plasma.If the IVS-II-654 mutation was not detected, we detected the SNP which linked to the IVS-II-654 mutation.Similarly, we used TT-FULL-COLD-PCR to enrich the CD41-42 mutations in the maternal plasma.At the same time, we used the conventional PCR to enrich CD41-42 mutation and IVS-II-654 mutation in the maternal plasma.RESULTS:(1) Nine cases of the SNP ( rs7480526) linked to the mutation at IVS-II-654 in HBB gene, and 11 cases of the SNP ( rs10768683) linked to the muta-tion at CD41-42 in HBB gene were detected.( 2 ) We detected 1 case who inherited the paternal β-thalassemia mutation (IVS-II-654).We did not directly detect patermal IVS-II-654 mutation in maternal plasma, but detected the SNP linked to the IVS-II-654 mutation in the other case and had 100%detection, and 2 cases inherited the paternal β-thalassemia muta-tions (CD41-42) in the maternal plasma by TT-FULL-COLD-PCR and had 100%detection.However, we detected nothing by conventional PCR.CONCLUSION:TT-COLD-PCR is applicable to enrich cell-free fetal DNA in maternal plasma and is a method in the field of noninvasive prenatal diagnosis.
ABSTRACT
Objective: To analyze the mutation status of K-ras gene in CRC (colorectal cancer) tissues and plasma samples of CRC patients, ultimately to promote the targeted agents against EGFR (epidermal growth factor receptor) (such as cetuximab and panitumumab, etc.) used in personalized treatment for CRC patients. Methods: The sequence of K-ras gene at specific sites was investigated in 431 CRC tissues and 23 plasma samples collected from 454 CRC patients, respectively. To improve the detection sensitivity, a combinatory approach was chosen which included the COLD-PCR (coamplification at lower denaturation temperature PCR) method, followed by DNA sequencing. Chi-square test was employed to analyze K-ras mutation frequencies in various sample types or subgroups of CRC patients according to age, and the difference was considered statistically significant if P value was less than 0.05. Results: The overall K-ras mutation rate was 25.29% in 431 CRC tissue samples. The two major forms of K-ras mutation were Gl 2D (mutation of glycine to an aspartate residue at codon 12) and Gl 3D (mutation of glycine to an aspartate residue at codon 13), and their occurrence frequencies were 12.99% and 6.26%, respectively. Interestingly, the overall K-ras mutation rate was 21.74% in blood samples from additional 23 CRC patients, without a significant difference from the mutation rate in the tumor samples (P > 0.05). Moreover, the occurrence frequency of anyone form of K-ras mutation was 37.94% in CRC patients aged 60 and over, which was significantly higher than the detection rate (7.30%) of the patients under the age of 60 (P < 0.01). Conclusion: COLD-PCR amplification combined with DNA sequencing method can be used to detect the mutation status of K-ras gene, and plasma samples can be used insteadly if CRC tumor tissues are unavailable. In addition, for elderly patients aged of 60 and over, it is suggested that K-ras mutation status should be routinely detected before treatment. Copyright © 2013 by TUMOR.