Download - Gene Therapy
Gene Therapy: The current solution for many dreadful diseases.
Soumyadip Das,Graduate student, Dept. of Biomedical Sc. & Engineering
Hanyang University, SeoulAdvisor: Dr. Suresh Ramakrishna.
Gene therapy:Gene therapy is the technology in which any gene, which is responsible for the development of a disease, is replaced with a healthy gene.
The gene therapy can be performed with the aim of gene editing accordingly,
A. Replacing a mutated gene that causes disease with a healthy copy of the gene.
B.Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
C.Introducing a new gene into the body to help to fight against a disease.
Gene Editing:The gene editing can be performed with the help of artificial programmable nucleases or in other words restriction enzymes, which produce site specific DNA-double strand break which leads to mutagenesis. Types of nucleases:I.Zinc finger nucleases (ZFN’s)
II.Transcription activator-like effector nucleases (TALENs)
III.RNA-guided engineered nucleases (RGENs)/CRISPR Cas9 system
Zinc-finger nucleases: (First generation nucleases)
Artificial restriction enzyme and have two domains;
DNA binding domain,genetically engineered to bind to specific site of DNA. It can recognise between 9-18 bp.
DNA cleaving domain,Fokl restriction enzymeused for cleavage of DNA.
Transcription-activator like effector nuclease (TALENs): Second generation nucleaseArtificial restrition enzyme generated by fusion of,
transcription activator (TAL) proteins or DNA binding domain
DNA cleaving domain (Fokl nuclease)
CRISPR/Cas9 (cluster regularly interspaced short palindromic repeat):Targeting RNA : CRISPR RNA Trans-CRISPR RNA
Cas9 protein: Which cleaves or break the double strands and induce a mutation.
Overlapped Extension PCR:
1. To Insert specific mutation at specific points in a sequence.
2.To splice (join) two smaller DNA fragments into larger polynucleotide.
After 1st PCR with Primers a+b and c+d,
We could see two fragments of definite sizes.
After 2nd PCR with primers a+d,
We could differentiate and understand whether the overlapping has happened or not, by determining the size of the band.
Kbp
20001000500
1.Transformation.2.Incubation overnight at 37 degree centigrade.3.Colonies.4.Screening those colonies (colony PCR).5.Positive colonies are Inoculated in growth media.6.Mini-prep of the Positive colonies. Send those for sequencing.