Tiêu đề Biotechnology Notes + Practice MCQS.pdf ✅

Biotechnology Dr. Muhammad Sohail, PhD 369 ➢ some of examples of vectors are, Plasmid, Lambda (λ) phage DNA, Yeast artificial chromosome, Cosmid (It is a combination of Plasmid and Phage DNA) etc. Steps for Integration of DNA (Gene of interest) insert into vector ➢ The integration of gene of interest into vector involves several following steps: 1 st step: ➢ The vector and gene of interest are digested by same restriction enzymes to create sticky ends. 2 nd step: ➢ The digested fragments are purified to isolate the desired size fragments. 3 rd step: ➢ The purified vector and DNA insert are ligated together through ligase. 4 th step: ➢ The ligated DNA is introduced into host cells through transformation using methods like electrophoresis, chemical transformation or heat shock method. ➢ The selected clones can be scaled up and used for various applications, including protein expression therapy or genetic modification. The techniques applied for the selection of vector ➢ The techniques which applied for selection of vector are based on the presence of suitable markers. ➢ The markers may be genetic elements like antibiotic resistance genes which produced different colors in host or fluorescent protein. Antibiotic resistance: ➢ AmpR gene, if present in plasmid the host can grow on a medium containing Ampicillin. Colour producing gene: ➢ LacZ gene contains vector if present in plasmid the host bacteria can hydrolyze X-Gal (a modified sugar) and produce blue color in bacteria which shows the presence of vector. ➢ When gene is inserted, the bacteria will not produce blue Colour. Fluorescent protein: ➢ Another approach is use of Gene florescent protein (GFP). The vector contains FPG with gene of interest, when vector with gene of interest is taken up by host cell the FPG also expresses and produces fluorescence which can be observed under fluorescence microscope. Polymerase Chain Reaction (PCR) ➢ Polymerase Chain Reaction (PCR) is a technique used for amplifying (cloning) a specific DNA fragment or gene into millions of copies. ➢ It takes place in vitro, in specific conditions and in the presence of enzymes. ➢ Following are the steps in gene replication through PCR: Denaturation: ➢ The first step in PCR is denaturing where the double stranded DNA template is heated to high temperature. i.e. 94-98oC to separate the two strands. Primer Annealing: ➢ The temperature then lowered abruptly to 50-56 oC and primer, a short DNA which is complementary to the ends of the target sequence are added. ➢ The primer anneals to the template DNA. ➢ It provides a starting point for DNA synthesis. Extension or polymerization: ➢ A heat stable DNA polymerase enzyme e.g., Taq polymerase, is added, along with a supply of nucleotides. ➢ The temperature is raised up to 72 oC which is the optimal temperature for DNA polymerase to extend the primer and synthesize a new complementary strand of DNA.