Corporate Banner
Satellite Banner
qPCR
Scientific Community
 
Become a Member | Sign in
Home>News>This Article
  News
Return

Cloning and Sequencing of the Light Chain Variable Region from NS-1 Myeloma

Published: Thursday, July 19, 2012
Last Updated: Thursday, July 19, 2012
Bookmark and Share
In this study, scientists synthesized 17 primers, 4 pairs of heavy chain primers and 9 light chain primers, to clone and sequence the genes encoding the variable region of the NS-1 cells using reverse transcription PCR (RT-PCR).

Abstract
The aim of this study was to clone the gene encoding the light chain variable region (V(L)) of the murine myeloma cell line P3/NS1/1-Ag4-1 (NS-1). Total RNA was prepared from the NS-1 cell line and its fusion hybridoma cell line 2F9. The V(L) gene was amplified using reverse transcription PCR (RT-PCR) with family-specific primer pairs. The PCR products were cloned into the pGEM(®)-T easy vector, transfected into E. coli DH5α cells and the positive recombinants were identified and purified with EcoRI digestion and agarose gel electrophoresis. The sequence was identified using an automatic DNA sequencer and analyzed online using the IMGT/V-QUEST program (version 3.2.21). The PCR product that was amplified with the P9 and P14 primers was approximately 392 bp in size. Following digestion with EcoRI, the band of interest was also detected in positive recombinants using agarose gel electrophoresis. The sequences from the NS-1 and 2F9 cells were identical. The whole sequence was 387 bp long, encoding 128 amino acids (AA), including a 60 bp leader sequence. There was a TAA stop codon at 385-387 bp and only one cysteine was found, at 112AA/128AA. Analysis using IMGT/V-QUEST determined the cloned V- and J-segments as murine IG(κ)KV3-12*01 and IG(κ)KJ2*01, respectively. Accordingly, the NS-1 V(L) gene belongs to the Ig(κ) gene family V3 subgroup. The NS-1 V(L) gene was successfully cloned and it is a pseudo-Igκ chain gene in NS-1 cells. This study may aid the sequencing of genes encoding monoclonal antibodies produced by mouse hybridomas raised with NS-1 myeloma.

This study is published online in Oncology Letters and is free to access.


Further Information

Join For Free

Access to this exclusive content is for Technology Networks Premium members only.

Join Technology Networks Premium for free access to:

  • Exclusive articles
  • Presentations from international conferences
  • Over 3,100+ scientific posters on ePosters
  • More than 4,500+ scientific videos on LabTube
  • 35 community eNewsletters


Sign In



Forgotten your details? Click Here
If you are not a member you can join here

*Please note: By logging into TechnologyNetworks.com you agree to accept the use of cookies. To find out more about the cookies we use and how to delete them, see our privacy policy.

Related Content

Selection of Reference Genes for Quantitative RT-PCR Analysis
The expression stability of 12 commonly used reference genes was analysed.
Thursday, April 02, 2015
Determination of Arabidopsis Thaliana Telomere Length by PCR
Researchers have designed two telomeric degenerated primers that amplify Arabidopsis telomeres by MMQPCR.
Friday, July 11, 2014
Methods for Multiplex Template Sampling in Digital PCR Assays
This article presents Multiplex Template Sampling, an alternative strategy to effectively increase template concentrations in dPCR analysis.
Wednesday, May 28, 2014
Detection of Large Expansions in Myotonic Dystrophy Type 1
The aims of this study were to verify the validity of triplet primed-PCR for the diagnosis of patients presenting with DM1 clinical findings and to simplify the testing procedure.
Monday, May 12, 2014
Genotoxicity Evaluation of Acephate and Profenofos by the PCR-RFLP Assay
The rampant use of organophosphorus insecticides has created a chemical environment which is proving harmful to the living system.
Thursday, April 24, 2014
An Alternative Suite of Universal Primers for Genotyping in Multiplex PCR
The new set of universal primers for multiplex genotyping were proven to be efficient and reliable in varied species.
Thursday, March 27, 2014
DNA Polymerase Preference Determines PCR Priming Efficiency
Researchers use high throughput sequencing to systematically demonstrate and characterize DNA polymerase priming bias.
Tuesday, February 11, 2014
MAR-Mediated Integration of Plasmid Vectors for In Vivo Gene Transfer and Regulation
The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models.
Thursday, December 05, 2013
Diagnostic Performance and Therapeutic Impact of PCR Assay in Patients with Suspected Sepsis
A rapid and accurate etiological diagnosis is essential in the successful management of patients with bloodstream infections.
Monday, November 25, 2013
Diabetic Retinopathy is Associated with Oxidative Stress and Mitigation of Gene Expression of Antioxidant Enzymes
The aim of the present study was to investigate different anthropometric and biochemical parameters among patients with DR and patients without DR.
Monday, October 07, 2013
Tool for the Identification of Targeted Sequences from Multidimensional High Throughput Sequencing Data
This article illustrated the use of InsertionMapper which consists of four independently working modules.
Monday, October 07, 2013
RNA-Seq Reveals Infection-Related Gene Expression Changes in Phytophthora capsici
This study provides a critical step to characterize the mechanisms of pathogenicity and virulence of P. capsici.
Thursday, September 12, 2013
Detection of Coinfection of Unusual Types of Bovine Papillomavirus by a PCR-RFLP Method
Findings point to the dissemination of BPVs with unclear pathogenic potential.
Tuesday, July 23, 2013
Indeterminate tcdB Using a C. difficile PCR Assay
C. difficile toxin RT-PCR is increasingly being adopted in clinical laboratories because it is more sensitive and reduces turnaround time when compared to toxin immunoassay.
Tuesday, July 23, 2013
Single-Reaction, Multiplex, RT-PCR for the Detection, Quantitation, and Serotyping of Dengue Viruses
Article demonstrates a diagnostic test that is more analytically sensitive than a commonly used reference molecular assay.
Tuesday, May 07, 2013
Scientific News
Finding Zika One Paper Disc At A Time
A novel, inexpensive method for detecting the Zika virus could help slow spread of outbreak, and potentially other future pandemic diseases.
Rapid Diagnosis of Bacterial Infections
Mass. General-developed compact system could shorten diagnostic time from days to hours, bring testing to point of care.
Monovar Drills Down Into Cancer Genome
Rice, MD Anderson develop program to ID mutations in single cancer cells.
Biomarkers for Profiling Prostate Cancer Patients
Exiqon A/S has announced the publication of validation of prognostic microRNA biomarkers for the aggressiveness of prostate cancer in independent cohorts.
Gene Analysis System Could Accelerate Pace of Research on the Space Station
The WetLab-2 system will enable spaceflight genomic studies involving a wide variety of biospecimen types in the unique microgravity environment of space.
Liquid Biopsy for NSCLC
'Liquid biopsy' blood test accurately detects key genetic mutations in most common form of lung cancer, study finds.
Rare DNA Will Have Nowhere To Hide
Two National Institutes of Health grants back Rice University effort to develop new diagnostics.
Seeing Cell to Cell Differences
Seeing differences between individual cells for first time explains symptoms of rare genetic disorders, finds penn study.
Circular RNAs Implicated in Cancer
Circular RNAs – like their protein counterparts – are also affected by genomic rearrangements in cancer, resulting in abnormal fusions.
Know Thy Helicobacter pylori
Non-invasive method for genotyping H.pylori developed.
Skyscraper Banner

SELECTBIO Market Reports
Go to LabTube
Go to eposters
 
Access to the latest scientific news
Exclusive articles
Upload and share your posters on ePosters
Latest presentations and webinars
View a library of 1,800+ scientific and medical posters
3,100+ scientific and medical posters
A library of 2,500+ scientific videos on LabTube
4,500+ scientific videos
Close
Premium CrownJOIN TECHNOLOGY NETWORKS PREMIUM FOR FREE!