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Articles by Timothy Block in JoVE
JoVE General
Kompleks Örnekleri spesifik protein Glikozilasyon arasında Multiplexed Yüksek verim Profilleme için Kimyasal-bloke Antikor Mikroarray
1Institute for Hepatitis and Virus Research, 2Department of Microbiology and Immunology, Thomas Jefferson University, 3Drexel University College of Medicine, 4Van Andel Research Institute, 5Institute for Hepatitis and Virus Research, Serome Biosciences Inc.
Bu çalışmada, özel proteinlerin glikozilasyon profil kullanılabilir lektin saptama yöntemi ile çoklanır yüksek verim antikoru mikroarray için geliştirilmiş bir protokol açıklanmaktadır. Bu protokol, yeni, güvenilir reaktif özellikleri ve önemli ölçüde daha önceki prosedüre göre zaman, maliyet ve laboratuar ekipmanları gereksinimlerini azaltır.
Other articles by Timothy Block on PubMed
A Locked Nucleic Acid Clamp-mediated PCR Assay for Detection of a P53 Codon 249 Hotspot Mutation in Urine
Hepatocellular carcinoma (HCC) has a 5-year survival rate of <10% because it is difficult to diagnose early. Mutations in the TP53 gene are associated with approximately 50% of human cancers. A hotspot mutation, a G:C to T:A transversion at codon 249 (249T), may be a potential DNA marker for HCC screening because of its exclusive presence in HCC and its detection in the circulation of some patients with HCC. A locked nucleic acid clamp-mediated PCR assay, followed by melting curve analysis (using the SimpleProbe), was developed to detect the TP53 249T mutation. In this assay, the locked nucleic acid clamp suppressed 10(7) copies of wild-type templates and permitted detection of 249T-mutated template, with a sensitivity of 0.1% (1:1000) of the mutant/wild-type ratio, assessed by a reconstituted standard within 2 hours. With an amplicon size of 41 bp, it detects target DNA sequences in short fragmented DNA templates. The detected mutations were validated by DNA sequencing analysis. We then tested DNA isolated from urine samples of patients with HCC for p53 mutations and identified positive TP53 mutations in 9 of 17 samples. The possibility of using this novel TP53 249T assay to develop a urine or blood test for HCC screening is discussed.
Methylation of the CpG Sites Only on the Sense Strand of the APC Gene is Specific for Hepatocellular Carcinoma
Hypermethylation of the promoter of the tumor suppressor gene, adenomatous polyposis coli (APC), occurs in various malignancies, including hepatocellular carcinoma (HCC). However, reports on the specificity of the methylation of the APC gene for HCC have varied. To gain insight into how these variations occur, bisulfite PCR sequencing was performed to analyze the methylation status of both sense and antisense strands of the APC gene in samples of HCC tissue, matched adjacent non-HCC liver tissue, hepatitis, cirrhosis, and normal liver tissues. DNA derived from fetal liver and 12 nonhepatic normal tissue was also examined. These experiments revealed liver-specific, antisense strand-biased CpG methylation of the APC gene and suggested that, although methylation of the antisense strand of the APC gene exists in normal liver and other non-HCC disease liver tissue, methylation of the sense strand of the APC gene occurs predominantly in HCC. To determine the effect of the DNA strand on the specificity of the methylated APC gene as a biomarker for HCC detection, quantitative methylation-specific PCR assays for sense and antisense strand DNA were developed and performed on DNA isolated from HCC (n = 58), matched adjacent non-HCC (n = 58), cirrhosis (n = 41), and hepatitis (n = 39). Receiver operating characteristic curves were constructed. With the cutoff value set at the limit of detection, the specificity of sense and antisense strand methylation was 84% and 43%, respectively, and sensitivity was 67.2% and 72.4%, respectively. This result demonstrated that the identity of the methylated DNA strand impacted the specificity of APC for HCC detection. Interestingly, methylation of the sense strand of APC occurred in 40% of HCCs from patients with serum AFP levels less than 20 ng/mL, suggesting a potential role for APC as a biomarker to complement AFP in HCC screening.
Detection of Hypermethylated Vimentin in Urine of Patients with Colorectal Cancer
We demonstrated previously that urine contains low-molecular-weight (LMW) (<300 bp), circulation-derived DNA that can be used to detect cancer-specific mutations if a tumor is present. The goal of this study was to develop an assay to detect the colorectal cancer (CRC)-associated, circulation-derived, epigenetic DNA marker hypermethylated vimentin gene (mVIM) in the urine of patients with CRC. An artificial 18-nucleotide DNA sequence was tagged at the 5' end of the primers of the first PCR cycle to increase the amplicon size, which was then integrated into the primers of the second PCR cycle. A quantitative MethyLight PCR-based assay targeting a 39-nucleotide template was developed and used to quantify mVIM in CRC tissues and matched urine samples. mVIM was detected in 75% of LMW urine DNA samples from patients with CRC (n = 20) and in 10% of urine samples of control subjects with no known neoplasia (n = 20); 12 of 17 LMW urine DNA samples (71%) but only 2 of 17 high-molecular-weight urine DNA samples (12%) from patients with mVIM-positive tissues contained detectable mVIM, suggesting that the mVIM detected in LMW urine DNA is derived from the circulation. The detection of mVIM in urine was significantly associated with CRC compared with controls (P < 0.0001, by Fisher's exact test). A potential urine test for CRC screening using epigenetic markers is discussed.
Impact of the Location of CpG Methylation Within the GSTP1 Gene on Its Specificity As a DNA Marker for Hepatocellular Carcinoma
Hypermethylation of the glutathione S-transferase π 1 (GSTP1) gene promoter region has been reported to be a potential biomarker to distinguish hepatocellular carcinoma (HCC) from other liver diseases. However, reports regarding how specific a marker it is have ranged from 100% to 0%. We hypothesized that, to a large extent, the variation of specificity depends on the location of the CpG sites analyzed. To test this hypothesis, we compared the methylation status of the GSTP1 promoter region of the DNA isolated from HCC, cirrhosis, hepatitis, and normal liver tissues by bisulfite-PCR sequencing. We found that the 5' region of the position -48 nt from the transcription start site of the GSTP1 gene is selectively methylated in HCC, whereas the 3' region is methylated in all liver tissues examined, including normal liver and the HCC tissue. Interestingly, when DNA derived from fetal liver and 11 nonhepatic normal tissue was also examined by bisulfite-PCR sequencing, we found that methylation of the 3' region of the promoter appeared to be liver-specific. A methylation-specific PCR assay targeting the 5' region of the promoter was developed and used to quantify the methylated GSTP1 gene in various diseased liver tissues including HCC. When we used an assay targeting the 3' region, we found that the methylation of the 5'-end of the GSTP1 promoter was significantly more specific than that of the 3'-end (97.1% vs. 60%, p<0.0001 by Fisher's exact test) for distinguishing HCC (n = 120) from hepatitis (n = 35) and cirrhosis (n = 35). Encouragingly, 33.8% of the AFP-negative HCC contained the methylated GSTP1 gene. This study clearly demonstrates the importance of the location of CpG site methylation for HCC specificity and how liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC.
