rDNA RECOrecombinant DNA technology, Assignments of Biopharmaceutics and Pharmacokinetics

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Recombinant DNA Technology & its

Applications

Dr. Bibhu Prasad Panda Associate Professor (Pharmaceutical Biotechnology) SPER Jamia Hamdard, New Delhi

Outlines of Presentation

• Steps Involved in r DNA Technology

• DNA Isolation and Analysis

• PCR Methods

• Restriction Enzymes & Vectors

• Recombinant Vector and Gene Transfer

• Screening of recombinant cell

• Application of r DNA Technology

Insulin, Interferon, Growth Hormones and HbSAg (vaccines)

DNA is the genetic material of most

organisms (from bacteria to humans)

Plasmid

Deoxyribonucleic acid (DNA) is a long

double-stranded chain of nucleotides

• DNA is the hereditary material passed on from generation to generation.
• DNA is made up of four nucleotides: A, C, G, and T.
• A always pairs with T.
• C always pairs with G.
• The two strands of DNA are in an antiparallel configuration.
• Two complementary DNA strands will separate when heated, and will spontaneously pair together again (hybridize) when cooled

Isolation of DNA

Analysis of DNA purity

• A 260/A280= 1.

If less than 1.8 =The DNA is impure

DNA Electrophoresis

A thermophilic (heat-loving) bacteria called

Thermus aquaticus is the source of Taq DNA

polymerase used in PCR reactions

Kary Mullis Invented the technique of polymerase chain reaction in the early 1980 Nobel Prize in 1993 Key technique used to make large quantities of DNA

The first round of PCR

A typical PCR protocol

 Begins with DNA containing a sequence to beamplified and a pair of synthetic oligonucleotide primers that flank the sequence.

 Next, denature the DNA to single strands at 94˚C.

 Rapidly cool the DNA (37-primers to complementary single strand65˚C) and anneal sequences flanking the target DNA.

 Extend primers at 70-75˚C using a heat- resistant DNA polymerase such as polymerase derived from Thermus aquaticus Taq****.

 Repeat the cycle of denaturing, annealing, andextension 20-45 times to produce 1 million (2 (^20) ) to 35 trillion copies (2^45 ) of the target DNA.

 Extend the primers at 70- allow incomplete extension products in the75˚C once more to reaction mixture to extend completely.  Cool to 4˚C and store or use amplified PCR product for analysis.

DNA cloning

• What Does It Mean: “To Clone”?

Clone: a collection of molecules or cells, all identical to an original molecule or cell

• To "clone a gene" is to make many copies of it - for example, by replicating it in a culture of bacteria.
• Cloned gene can be a normal copy of a gene (= “wild type”).
• Cloned gene can be an altered version of a gene (= “mutant”).
• Recombinant DNA technology makes manipulating genes possible.

Basics of type II Restriction

Enzymes

• No ATP requirement.

• Recognition sites in double stranded DNA

have a 2-fold axis of symmetry – a

“palindrome”.

• Cleavage can leave staggered or "sticky"

ends or can produce "blunt” ends.

Recognition/Cleavage Sites of

Type II Restriction Enzymes

Cuts usually occurs at a palindromic sequence

Sma I: produces blunt ends

5 ´ CCCGGG 3´

3 ´ GGGCCC 5´

Eco RI: produces sticky ends 5 ´ GAATTC 3´

3 ´ CTTAAG 5´