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Analysis of TRG Gene Rearrangement by Southern Blot Technology (CAT#: STEM-MHT-0003-LGZ)

Introduction

Official Full Name: T cell receptor gamma locus<br />Also known as: TCRG; TRG@<br />T cell receptors recognize foreign antigens, which are processed into small peptides and bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs). Each T cell receptor is a dimer consisting of one chain and one chain or one chain and one chain. In a single cell, T cell receptor loci are rearranged and expressed in the order delta, gamma, beta, and alpha. If both delta and gamma rearrangements result in a functional chain, the cell expresses delta and gamma. If not, the cell rearranges the beta and alpha loci. This region represents the germline organization of the T cell receptor gamma locus. The gamma trace consists of V (variable), J (joint) and C (constant) segments. During T cell development, the γ chain is synthesized through recombination events at the DNA level, linking the V segment with the J segment; the C segment is subsequently joined by splicing at the RNA level. Recombination of many different V segments with a few J segments provides broad antigen recognition. Additional diversity is junctional diversity obtained by random addition of nucleotides by terminal deoxynucleotidyl transferase. Several V segments of the gamma locus are known not to encode proteins and are considered pseudogenes. Somatic rearrangements of the gamma locus were observed in T cells from patients with T-cell leukemia and ataxia telangiectasia.




Principle

Under certain conditions, two single strands of nucleic acid with certain homology can be specifically hybridized to form double strands according to the principle of base complementarity. Generally, DNA molecules to be detected are digested with restriction enzymes, separated by agar-gel electrophoresis, denatured and transferred to nitrocellulocellulose film or nylon film or other solid phase support according to their position in the gel, fixed and then reacted with DNA probes labeled with isotopes or other markers. This is followed by autoradiography or an enzyme reaction to detect the amount of specific DNA molecules. If the object to be tested contains a sequence that is complementary to the probe, the two are combined by the principle of base complementarity, and the free probe is washed and detected by self-development or other suitable techniques, thus revealing the fragment to be tested and its relative size.

Applications

Gene Rearrangement Detection

Procedure

1. Sample Processing
2. DNA Extraction and Digestion
3. Gel Electrophoresis
4. Gel Pretreatment
5. Transfer membrane
6. Probe Labeling
7. Prehybridization (blocking)
8. Southern hybridization
9. Membrane washing
10. Autoradiographic Assay
11. Results Analysis

Materials

Sample: DNA, Bacterial Fluid/Tissue/Cell