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:6/24/2016)... , ... June 24, 2016 , ... Researchers at the ... commonly-identified miRNAs in people with peritoneal or pleural mesothelioma. Their findings are the subject ... it now. , Diagnostic biomarkers are signposts in the blood, lung fluid or ...
(Date:6/23/2016)... Mass. , June 23, 2016   ... development of novel compounds designed to target cancer ... napabucasin, has been granted Orphan Drug Designation from ... the treatment of gastric cancer, including gastroesophageal junction ... stemness inhibitor designed to inhibit cancer stemness pathways ...
(Date:6/23/2016)... 2016 Houston Methodist Willowbrook Hospital has ... Association to serve as their official health care ... Willowbrook will provide sponsorship support, athletic training services, ... coaches, volunteers, athletes and families. "We ... Association and to bring Houston Methodist quality services ...
(Date:6/23/2016)... , June, 23, 2016  The Biodesign Challenge (BDC), ... new ways to harness living systems and biotechnology, announced ... (MoMA) in New York City . ... participating students, showcased projects at MoMA,s Celeste Bartos Theater ... Antonelli , MoMA,s senior curator of architecture and design, ...
Breaking Biology Technology:
(Date:6/1/2016)... NEW YORK , June 1, 2016 ... Biometric Technology in Election Administration and Criminal Identification to ... According to a recently released TechSci Research report, " ... Sector, By Region, Competition Forecast and Opportunities, 2011 - ... $ 24.8 billion by 2021, on account of growing ...
(Date:5/20/2016)... MINNEAPOLIS , May 20, 2016  VoiceIt ... technology partnership with VoicePass. By working ... user experience.  Because VoiceIt and VoicePass take slightly ... two engines increases both security and usability. ... expressed excitement about this new partnership. ...
(Date:5/12/2016)... DALLAS , May 12, 2016 ... has just published the overview results from the Q1 ... of the recent wave was consumers, receptivity to a ... wearables data with a health insurance company. ... choose to share," says Michael LaColla , CEO ...
Breaking Biology News(10 mins):