Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.
Few studies have examined Chinese patients with chronic hepatitis B who exhibit hepatitis B surface antigen (HBsAg) seroclearance. We comprehensively studied the biochemical, virological, histological, and clinical aspects of 92 patients with HBsAg seroclearance (median follow-up, 126 months). Ninety-two HBsAg-positive controls matched for age, sex, and duration of follow-up were also recruited. Liver biochemistry, serum hepatitis B virus (HBV) DNA levels, and development of clinical complications were monitored. Intrahepatic total and covalently closed circular (ccc) HBV DNA were measured quantitatively in 16 patients. HBV genotype was determined in 30 patients. The mean age at HBsAg seroclearance was 48.8 (؉ 13.81) years. There was a significant improvement in serum alanine aminotransferase levels after HBsAg seroclearance (p<0.0001). Patients with genotype B had a higher chance of HBsAg seroclearance than those with genotype C (P ؍ .014). Ninety-eight percent of patients had undetectable serum HBV DNA. Thirty-seven percent of patients had low titer of intrahepatic HBV DNA, mainly in the form of cccDNA (71%-100%). All 14 patients with liver biopsies had near normal histology. There was no difference in the risk of development of hepatocellular carcinoma (HCC) between patients with and without HBsAg seroclearance. However, the mean age of HBsAg seroclearance was significantly older in patients with HCC than in patients without HCC (P ؍ .016). In conclusion, patients with HBsAg seroclearance had favorable biochemical, virological, and histological parameters. Intrahepatic HBV DNA level was low and predominantly in the form of cccDNA. However, HCC could still develop, particularly in patients with cirrhosis who had HBsAg seroclearance at an older age. (HEPATOLOGY 2004;39:1694 -1701.) H epatitis B surface antigen (HBsAg) seroclearance is a rare event in Chinese patients with chronic hepatitis B virus (HBV) infection who acquire the disease early in life. The estimated annual incidence of HBsAg seroclearance is 0.1% to 0.8%. 1,2 Undetectable HBsAg in the serum is usually due to a decrease in viremia rather than the emergence of HBsAg mutants. 3 Some studies demonstrate a favorable outcome in terms of histological features and development of hepatocellular carcinoma (HCC). 2,4 However, these findings are not confirmed by other studies. 5,6 The presence of HBV DNA within the liver in patients with HBsAg seroclearance has been demonstrated in some studies, 7-9 but it is not known whether the intrahepatic HBV DNA is replicative or nonreplicative-that is, in the form of covalently closed circular (ccc) DNA. Also, it is uncertain how much residual virus is in extrahepatic reservoirs like peripheral blood mononuclear cells (PBMC).Therefore, we sought to determine the factors associated with and the significance of HBsAg seroclearance in Chinese HBV patients by examining the following parameters: (1) liver biochemistry, (2) HBV DNA in the serum and PBMC, (3) HBV genotypes, (4) intrahepatic total and cccDNA, (5) liver h...
The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10 7 reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.
This study examined a signal amplification assay, the Invader assay, for the quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in liver biopsies and sera. DNA was extracted from liver biopsy and serum samples were collected from 16 hepatitis B e antigen (HBeAg)-positive and 36 antibody-to-HBeAg-positive (anti-HBe-positive) chronic hepatitis B patients. The amount of total HBV DNA and cccDNA was measured using the Invader assay. Anti-HBe-positive patients had lower median total intrahepatic HBV DNA (P < .001) and intrahepatic cccDNA levels (P ؍ .001) than HBeAg-positive patients. Intrahepatic cccDNA correlated positively with the total intrahepatic HBV DNA (r ؍ 0.950, P < .001). However, the proportion of intrahepatic HBV DNA in the form of cccDNA was inversely related to the amount of total intrahepatic HBV DNA (r ؍ ؊0.822, P < .001). A small amount of cccDNA was detected in 39 of 52 (75%) serum samples. Anti-HBe-positive patients had lower median serum cccDNA levels than HBeAg-positive patients (P ؍ .002). Serum HBV DNA correlated positively with intrahepatic total HBV DNA (r ؍ 0.778, P < .001) and intrahepatic cccDNA (r ؍ 0.481, P ؍ .002). In conclusion, the Invader assay is a reliable assay for the quantitation of cccDNA. vitro studies have shown that lamivudine has a profound effect on relaxed circular DNA (rcDNA) while having little or no effect on cccDNA. 2,3 This is one possible reason for the rebound of HBV DNA to pretreatment levels often seen after lamivudine withdrawal. 4 Another nonreplicative form of HBV DNA is the double-stranded linear (DL) form produced by in situ priming during HBV replication. 5 DL DNA is a possible precursor to HBV DNA integration. 6 -8 It can also form cccDNA through nonhomologous recombination at its ends via a process called illegitimate replication. 9,10 cccDNA monitoring and the development of an accurate quantitative assay for cccDNA are becoming important in the understanding of the natural history and management of chronic hepatitis B (CHB). Most attempts for the quantitation of cccDNA have been made with liver biopsies from ducks or woodchucks. [11][12][13][14][15][16] Quantitation of cccDNA in human peripheral blood mononuclear cells and liver biopsies has been performed. [17][18][19][20] In these studies, primers spanning across the gap in the minus strand and corresponding to the variable region on the plus strand were used to amplify across noninterrupted cccDNA. It should be noted that, even with selective polymerase chain reaction (PCR) methods,
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