Navigation Links
Detection of Mutant K-ras in a Kindred With Hereditary Pancreatic Cancer by DGGE

David A. Crispin,* Ru Chen,* Michael B. Kimmey,** Teresa A. Brentnall**
* Departments of Pathology and
**Medicine, Division of Gastroenterology, University of Washington, Seattle, Washington.


Introduction

Pancreatic cancer is the fourth leading cause of cancer death in the U.S. and its frequency is rising. At the time of diagnosis, 9699% of patients are incurable and will shortly die. Current methods to evaluate patients for pancreatic adenocarcinoma include Endoscopic Retrograde Cholangiopancreatography (ERCP), abdominal CT, ultrasound, and serum markers. These methods of diagnosis are often insensitive or equivocal in early disease. Tumorigenesis is believed to involve the K-ras oncogene, DCC, p16, APC, bcl-2 and p53 tumor suppressor genes; screening for mutations or loss of heterozygosity in these genes may provide better diagnostic tests that are more sensitive and specific.1 The study of families in which cancer is inherited in an autosomal dominant fashion has provided considerable insight into understanding the molecular basis for pancreatic cancer. We have previously reported an extensive kindred in which pancreatic cancer is inherited in an autosomal dominant fashion and is associated with development of pancreatic insufficiency prior to the diagnosis of cancer.2 We have utilized the DCode system for denaturing gradient gel electrophoresis (DGGE) to identify patient samples with K-ras mutations.


Materials and Methods

Genomic DNA was isolated from tissue or from fluid obtained from patients at ERCP. K-ras exon 1 was amplified from genomic DNA using a thermal cycler and primers as described by Imai et al.3 Amplifications were carried out using 300 ng of DNA template in a buffer containing 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3, with 2 units of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 200 M dNTPs, and 15 pmol of each primer in a total volume of 50 liters. Reactions were denatured for 3 minutes at 95 C, which was followed by thirty seven cycles with the following profile: 95 C (20s), 55 C (60s), 72 C (40s). The 110 bp product was run on a 3% agarose gel and visualized using ethidium bromide staining.

Successful samples were run on a 10% acrylamide 080% perpendicular DGGE gel at 150 V for 2 hours at 56 C, then stained with ethidium bromide to find the optimal conditions for parallel DGGE (Figure 1). This was determined to be 30%60%. Samples were then run on a parallel DGGE gel at 56 C for 4 to 5 hours at 150 V, and stained with ethidium bromide. Positive samples were sequenced using dye terminator chemistry and run on an ABI Prism (Perkin Elmer).


Results

Exon 1, containing codons 12 and 13, of the K-ras gene was examined for the presence of k-ras mutations by DGGE. Constitutional and pancreatic tissues or pancreatic juice in eight of the family members were evaluated. All samples that were positive by DGGE (Figure 2) were confirmed by DNA sequencing. Three of these individuals had K-ras mutations in codon 13 present in pancreatic cancer or precancerous tissue and three had mutations in codon 12. There was no evidence of K-ras mutation in the metastatic pancreatic cancer tissue from individual III.19, even when DNA was subcloned, or from the ERCP fluid or dysplastic tis sue from III.15 (Table 1).


Discussion

K-ras mutations are a common event in pancreatic adenocarcinoma. Screening for K-ras mutations along with a panel of other markers may prove useful in early diagnosis of this disease. DGGE is a practical tool in screening samples for the presence of such mutations.


References

1. Rozenblum, E., Schutte, M., Goggins, M., Hahn, S. A., Panzer, S., Zahurak, M., Goodman, S. N., Sohn, T. A., Hruban, R. H., Yeo, C .J. and Kern, S. E., Cancer Res., 57(9), 17311734 (1997).

2. Evans, J. P., Burke, W., Chen, R., Bennett, R. L., Schmidt, R. A., Dellinger, E. P., Kimmey, M., Crispin, D., Brentnall, T. A. and Byrd, D. R., J. Med. Genet., 32(5), 330335 (1995).

3. Imai, M., Tomokazu, H. and Ogawa, K., Cancer, 73(11), 27272733 (1991).


back to top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. A New PCR-based Mycoplasma Detection Method
3. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
4. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
5. Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes
6. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
7. The DIG System Nonradioactive and Highly Sensitive Detection of Nucleic Acids
8. Quantitative Measurement of Cell Proliferation Using the BrdU ELISA: A Comparison Between Colorimetric and Chemiluminescent Detection
9. Quantification of Nucleosomes in Serum by the Cell Death Detection ELISAplus
10. A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
11. In Situ Cell Death Detection Kit
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... , ... June 27, 2016 , ... Parallel 6 ... trials, announced today the Clinical Reach Virtual Patient Encounter CONSULT module which ... with the physician and clinical trial team. , Using the CONSULT module, patients and ...
(Date:6/27/2016)... Ginkgo Bioworks , a leading organism design company ... as one of the World Economic Forum,s Technology ... companies. Ginkgo Bioworks is engineering biology to manufacture ... the nutrition, health and consumer goods sectors. The ... Fortune 500 companies to design microbes for their ...
(Date:6/24/2016)... ... June 24, 2016 , ... Researchers at the Universita Politecnica delle ... people with peritoneal or pleural mesothelioma. Their findings are the subject of a new ... , Diagnostic biomarkers are signposts in the blood, lung fluid or tissue of mesothelioma ...
(Date:6/23/2016)... , June 23, 2016 /PRNewswire/ - FACIT has ... Ontario biotechnology company, Propellon Therapeutics Inc. ... and commercialization of a portfolio of first-in-class WDR5 ... targets such as WDR5 represent an exciting class ... in precision medicine for cancer patients. Substantial advances ...
Breaking Biology Technology:
(Date:4/28/2016)... and BANGALORE, India , April 28, 2016 ... a product subsidiary of Infosys (NYSE: INFY ), ... a global partnership that will provide end customers ... mobile banking and payment services.      (Logo: ... innovation area for financial services, but it also plays a ...
(Date:4/26/2016)... , April 27, 2016 ... the  "Global Multi-modal Biometrics Market 2016-2020"  report to ... ) , The analysts forecast the ... CAGR of 15.49% during the period 2016-2020.  ... number of sectors such as the healthcare, BFSI, ...
(Date:4/15/2016)... , April 15, 2016 ... "Global Gait Biometrics Market 2016-2020,"  report to their ... ) , ,The global gait biometrics market ... 13.98% during the period 2016-2020. Gait ... which can be used to compute factors that ...
Breaking Biology News(10 mins):