Navigation Links
Detection of Mutations in the Fas Antigen of Lymphoma Tumors by RT-PCR and Denaturing Gradient Gel Electrophoresis

Terry H. Landowski, Ibrahim Buyuksal, and William S. Dalton H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa FL 33612


Introduction

Identifying of mutations within specific genes has become an important strategy in determining the diagnosis and prognosis for many diseases, including cancer. Several techniques have been developed to examine heterogeneous tissue samples for specific mutations, including restriction fragment length polymorphism (RFLP), single stranded conformation polymorphism (SSCP), and denaturing gradient gel electrophoresis (DGGE), among others. These techniques are useful for rapid screening of heterogeneous tissues, and each has its individual strength.1 Using RT-PCR and DGGE, we have examined the cytoplasmic region of the Fas antigen known as the death domain in tumor specimens from patients with lymphoma of various histiocytic origins. These mutations may contribute to the failure of the tumor cells to undergo apoptosis, and thus contribute to the pathogenesis and progression of the disease.2


Materials and Methods

Tumor biopsies were obtained during postoperative resection, and peripheral blood lymphocytes for controls were obtained from normal volunteers. Total RNA was isolated from cryopreserved specimens and 50-100 ng of RNA was reverse transcribed by oligo dT priming. PCR primers for the FasIII region were as previously described2 with the addition of a 42 bp GC clamp on the forward primer.3 Following 35 cycles of amplification in a 9600 Perkin Elmer thermocycler, the PCR products were denatured for 5 minutes at 95 C and reannealed for 1 hour at 55 C to allow heteroduplex formation.

Melting temperature of the 430 bp PCR product was predicted using MacMelt software, and confirmed by analysis on a 6% acrylamide gel (37.5:1) with a perpendicular gradient of 12.537.5% denaturant. Electrophoresis in the DCode system was at a constant temperature of 60 C in 1x TAE buffer. Gels were run at 100 V for 2.5 hours. Optimum strand separation occurred at 2324% denaturant, which correlated to the predicted melting temperature of 6570 C. All further analyses were done in a parallel gradient of 1530% denaturant at 60 C, with running time extended to 4.5 hours. PCR products were detected by ethidium bromide staining.


Results and Discussion

Hematological tumors typically contain a heterogeneous population of cells that includes both neoplastic and normal cells. The identification of tumor specific mutations requires a method that can separate mutant sequences from normal sequences for isolation and identification. Sensitivity of the techniques is important in diseases such as lymphoma, which typically contain large numbers of normal stromal cells and infiltrating lymphocytes.

To determine the sensitivity of the DGGE analysis, we took advantage of a previously identified polymorphism in the Fas antigen, in which bp836 is changed from C to T.4 Direct sequencing of PCR products has shown that the T cell leukemia cell line CEM is heterozygous for the alternative codon, ACT, while the myeloma cell line 8226 expresses only the higher frequency codon, ACC. RNA was isolated from CEM and 8226 cell lines, and titrated at 1%, 5%, 10%, 30%, and 50% to determine the minimal amount of polymorphic cDNA detectable (Figure 1). Using ethidium bromide staining, we were able to easily identify 5% polymorphic RNA, and even as little as 1% was detectable.

Tumor specimens from patients with lymphoma were examined for mutations in the signal transducing domain of the Fas antigen by RT-PCR and DGGE analysis. Figure 2 shows a representative ethidium stained DGGE gel including 10 patient samples. The CEM T cell line serves as a positive control, and demonstrates the heteroduplex bands formed by polymorphic and wild type cDNA. The myeloma cell line 8226, which serves as a negative control, has been fully sequenced and shown to be wild type sequence. Of the patient specimens demonstrated in Figure 2, two display a single band characteristic of wild type sequence. Patient specimens 4, 5, 7, 9, and 10 demonstrate heteroduplex band formation, indicating an alteration in the cDNA sequence. These bands were extracted from the gel and reamplified by PCR for direct sequencing. In addition, the altered mobility of the single band seen in patient 8 is suggestive of a homozygous mutation. This band was also extracted for sequence analysis.

With the high frequency of polymorphisms reported at bp 836 in the Fas antigen, we sought to develop a screening method to distinguish the known polymorphism from other unknown mutations that could have functional significance. Expression of the alternative codon, ACC, introduces a unique restriction site for the enzyme DraI which is not present in the wild type cDNA sequence. Cleavage of the FasIII cDNAs with DraI results in an 368 bp fragment when the polymorphic codon is expressed, while this enzyme does not cut the wild type cDNA. Patient samples that demonstrated heteroduplex formation on DGGE analysis were incubated overnight at 37 C with 0.5U DraI, denatured for 5 minutes at 95 C, and allowed to reanneal at 55 C for 1 hour. Digested products were loaded on the DGGE acrylamide gel, and electrophoresed 3.5 hours under conditions described above (Figure 3). This treatment resulted in the disappearance of one of the heteroduplex bands in specimens with the polymorphism and the appearance of a truncated homoduplex band (see lanes 1, 4, 5, 8, and 9 of Figure 3). Patient specimens that are homozygous for the polymorphism display only the truncated homoduplex (lane 10), while patient specimens with mutations in addition to the polymorphism retained two heteroduplex bands (lanes 7 and 11).


Conclusions

Crosslinking of the Fas antigen by antibody or specific ligand induces apoptosis in susceptible cells. Mutations within the signal transducing domain may render the cells resistant to programmed cell death, and contribute to the progression of neoplastic disease. We have utilized the methods of RT-PCR and DGGE to detect small changes in the coding sequence of the Fas antigen in hematological tumors.

In contrast to many other rapid screening techniques, DGGE is based on known physical parameters of a particular gene. By calculating the predicted melting temperature of a segment of DNA, analytical conditions can be optimized for maximum accuracy and sensitivity.5 We have demonstrated that DGGE can be used to detect as little as 1% of total RNA containing a single base change with non-radioisotopic techniques. In addition, with a combination of restriction digest and DGGE analysis, we have developed a technique that can be used to distinguish between previously characterized non-coding changes in the Fas antigen, and unknown mutations. Identification of the known polymorphism reduces the number of bands which must be fully sequenced, and contributes to the overall efficiency of the genetic screening.


References

1. Eng, C. and Vijg, J. Nature Biotech., 15, 422426 (1997).

2. Landowski, T. H., Qu, N., Buyuksal, I., Painter, J. S. and Dalton, W. S., Blood (1997).

3. Sheffield, V. C., Cox, D. R., Lerman, L. S. and Myers, R. M. Proc. Natl. Acad. Sci. USA, 86, 232236 (1989).

4. Fiucci, G. and Ruberti, G. Immunogen., 39, 437439 (1995).

5. Myers, R. M., Maniatis, T. and Lerman, L. S., Meth. Enz., 155, 501527 (1987).


The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-LaRoche. Use of the PCR process requires a license.


back to top
'"/>

Source:


Page: All 1 2 3 4 5

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:
(Date:9/2/2015)... Research and ... addition of Jain PharmaBiotech,s new report ... to their offering. The scope ... in situ hybridization (FISH), comparative genomic hybridization ... application of nanobiotechnology, microarrays, real-time polymerase chain ...
(Date:9/2/2015)... ... September 02, 2015 , ... Drug companies increasingly are adopting ... Research reveals in its new report that the U.S. will generate the greatest ... generates the most market demand. , BCC Research examines plant-derived drugs in ...
(Date:9/2/2015)... 2015 Australian prostate cancer technology company Minomic ... prize recognising ,outstanding, science that uses its patented antibody. ... a novel prostate cancer diagnostic test known as MiCheck® ... taking out the peer-reviewed Australian Museum Eureka Science Prize ... This prize is awarded for ground breaking research ...
(Date:9/2/2015)... , Sept. 2, 2015 Veracyte, Inc. ... H. Anderson , president and chief executive officer, will ... Wednesday, September 16, 2015 at 10:30 a.m. ET in ... The live audio webcast and subsequent replay may ... webcast will be available shortly after conclusion of the ...
Breaking Biology Technology:Global Cytogenetics Technologies Study 2015-2025 - Latest Markets and Companies Analysis 2Global Cytogenetics Technologies Study 2015-2025 - Latest Markets and Companies Analysis 3Plant-derived Compounds Poised to Assume Greater Role in Global Drug Markets, According to BCC Research 2Plant-derived Compounds Poised to Assume Greater Role in Global Drug Markets, According to BCC Research 3Prostate Cancer Company Minomic Wins Prestigious Eureka Science Prize 2Prostate Cancer Company Minomic Wins Prestigious Eureka Science Prize 3Veracyte to Present at Morgan Stanley Global Healthcare Conference 2
... Group of representatives,from seven international health-care organizations call ... patients with hypertension, the single,most important cause of ... working group to identify 5 specific concrete practical ... of the Journal of Human,Hypertension(1): Detect and Prevent ...
... Research Will Create Jobs, Attract ... ... government is providing $150,million through the Next Generation Jobs Fund to ... John Wilkinson has announced., "Attracting these investments will create good-paying ...
... Portfolio, - Conference Call Scheduled for 5 February 2008, ... ... BRISBANE, Australia, Feb. 4 Progen,Pharmaceuticals Limited (ASX: PGL; ... Agreement to acquire privately-held U.S.,oncology company CellGate, Inc. The CellGate ...
Cached Biology Technology:Experts and Representatives From Leading International Health-Care Organizations Call to Stop the Hypertension Pandemic, a Worldwide Silent Killer 2Experts and Representatives From Leading International Health-Care Organizations Call to Stop the Hypertension Pandemic, a Worldwide Silent Killer 3Experts and Representatives From Leading International Health-Care Organizations Call to Stop the Hypertension Pandemic, a Worldwide Silent Killer 4Experts and Representatives From Leading International Health-Care Organizations Call to Stop the Hypertension Pandemic, a Worldwide Silent Killer 5Sparking Growth In Ontario's Biopharmaceutical Industry 2Sparking Growth In Ontario's Biopharmaceutical Industry 3Progen Expands Drug Development Pipeline Through Acquisition of CellGate 2Progen Expands Drug Development Pipeline Through Acquisition of CellGate 3Progen Expands Drug Development Pipeline Through Acquisition of CellGate 4Progen Expands Drug Development Pipeline Through Acquisition of CellGate 5
(Date:8/24/2015)... 2015 The consulting company Frost & ... manufacturer DERMALOG and its customized solutions and products for ... of the Year Award". DERMALOG is particularly successful with a ... -Cross reference: Picture is available at AP Images ... On Thursday evening, in South Africa,s ...
(Date:8/20/2015)... -- NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or ... growing mobile commerce market and creator of the Wocket® ... Alliance and the EMV Migration Forum.  ... Alliance Latin America (SCALA) Chapter provides NXT-ID, Inc. direct ... promoting adoption of smart card technology. To ...
(Date:8/17/2015)... Conn. , Aug. 17, 2015  NXT-ID, Inc. ... biometric authentication company focused on the growing mobile commerce ... its new sales campaign for Q4 2015. The new ... marketing team consisting of Amplitude Marketing Group ( http://amplitudemarketing.com/ ... Marketing Group is a leader in retail driven marketing ...
Breaking Biology News(10 mins):Frost & Sullivan Honors DERMALOG with Biometrics Prize for Achievements in Africa 2Frost & Sullivan Honors DERMALOG with Biometrics Prize for Achievements in Africa 3NXT-ID, Inc. Joins Smart Card Alliance and EMV Migration Forum 2NXT-ID, Inc. Joins Smart Card Alliance and EMV Migration Forum 3NXT-ID, Inc. Joins Smart Card Alliance and EMV Migration Forum 4NXT-ID, Inc. Launches Sales Campaign for Wocket Smart Wallet with Award Winning Marketing Team 2NXT-ID, Inc. Launches Sales Campaign for Wocket Smart Wallet with Award Winning Marketing Team 3
... intensely interested in telomeres, the protective caps on the ... UC Santa Cruz used a novel technique to reveal ... guide the development of new anti-cancer drugs. ... of chromosomes that serve a protective function analogous to ...
... Dietary changes since the early 1960s have fueled a sharp ... the food consumed by the average person over the course ... researchers at McGill University. Between 1961 and 2007, rising ... in the world,s per capita "phosphorus footprint," the researchers conclude ...
... ANN ARBORForty master,s and professional-degree students from eight schools ... the Dow Sustainability Fellows Program today, marking the first ... last spring. In total, the fellows will ... becoming part of a collaborative community of sustainability scholars ...
Cached Biology News:Novel technique reveals dynamics of telomere DNA structure 2Novel technique reveals dynamics of telomere DNA structure 3Dietary shifts driving up phosphorus use 240 U-M students named Dow Sustainability Fellows 2
... Simultaneously perform 96 assays on four slides. ... microarray slides with 96 (6mm x 7mm) ... gene expression, or screening analysis studies. Unlike ... fit gasket, eliminating the presence of contaminating ...
... of Use : GeneTraffic is built to ... biological research. GeneTraffic provides visual and computational ... data prior to analysis. , Data ... projects using the MIAME (Minimum Information About ...
... siTrio siRNA --- a cocktail of 3 ... of interestis our most popular product. The ... 75% knockdown of your target gene when ... for transfection and confirmation of optimal transfection ...
... kit #27295 has improved stability and convenience from ... not only has overcome this problem but also ... have changed the formulation of HRP conjugated antibody ... By this change, the stability of the conjugate ...
Biology Products: