Recombinant DNA Technology

Presented by

Gayathri S Mohan

DNA- the genetic secret!!

Encodes the genetic instructions of all known living organisms and many viruses.Nucleotides are the basic building block. Nucleotide= Sugar +

phosphate + Nitrogen bases.

4 Nitrogen basesAnti-parallel strands

Nitrogen bases

• Adenine (A)• Guanine (G)

• Thymine (T)• Cytosine( C)

found in pairs, with A & T and G & C Double helixsequence and number of bases creates the diversity

DNA mRNA Proteins

purines

 pyrimidines

What is Gene???

• A gene is a stretch of DNA that codes for a type of protein that has a function in the organism.

• It is a unit of heredity in a living organism.. All living things depend on genes

• Genes hold the information to build and maintain an organism's cells and pass genetic traits to offspring.

Recombinant DNA Technology

Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other, which can replicate in the living cell.

Recombinant DNA is also called Chimeric DNA Developed by Boyer and Cohen in 1973 3 different methods of DNA recombination • Transformation• Non-bacterial Transformation• Phage induction

Recombinant DNA Technology          

Basic steps involved in recombinant DNA technology 

Isolation of the gene of interest

Preparation of Vector DNA and DNA to be cloned

Insertion of the gene to the vector molecule and ligation

Introduction of the vector DNA to the appropriate host cell

Amplification of the recombinant DNA molecule in host cell.

Overview of rDNA technologyBacterial cell

DNA containinggene of interest

Bacterialchromosome Plasmid

Isolate Plasmid Gene of interest

Enzymatically cleaveDNA into fragments.

Isolate fragment with the gene of interest.

Insert gene into plasmid.

Insert plasmid and gene into bacterium.

Culture bacteria.

But let’s look at it in more detail....

Isolation of gene DNA molecule is extracted from the cell by using cell lysing method

Homogenization Centrifugation

Gene of interest is isolated using probes and electrophoresis

DNA which is to be cloned have to be inserted in to a vector molecule which act as a carrier of the DNA to the host cell.

The choice of a vector depends on the design of the experimental system and how the cloned gene will be screened or utilized subsequently.

Commonly used vectors are Plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), yeast 2 micron plasmid, retrovirus, baculovirus vector

Plasmid vector

 (antibiotic resistance gene, such as ampr and tetr

Covalently closed, circular, double stranded DNA molecules that occur naturally and replicate extra chromosomally in bacteria and in some fungi.

Eg: pBR 322 and pUC-18

characteristic of an ideal plasmid

(i)Presence of minimum amount of its own DNA.

(ii) Recognition sites for restriction endonuclease

(iii)Presence of at least two markers with recognition site being present in one of the two markers

(iv)Relaxed replication control so that the recombinant plasmid is capable of forming several copies.

A plasmid containing resistance to an antibiotic (usually ampicillin) or Tetracycline, is used as a vector.

Restriction Endonucleases

Important tool for rDNA technology is the Restriction Enzymes

Bacterial enzymes that cut DNA molecules only at restriction sites Molecular scissors Palindromic sequences are the recognition siteseg: EcoRI with recognition site GAATTC

5´ GAATTC 3´ 3´ CTTAAG 5

Categorized into two groups based on type of cut• Cuts with sticky ends• Cuts with blunt ends if one strand extends beyond the complementary region, then the DNA is

said to possess an overhang and it will have sticky ends.

Commonly used restriction enzymes

• EcoRI – Escherichia coli strain R, 1st enzyme• BamHI – Bacillus amyloliquefaciens strain H, 1st enzyme• DpnI – Diplococcus pneumoniae, 1st enzyme • HindIII – Haemophilus influenzae, strain D, 3rd enzyme• BglII – Bacillus globigii, 2nd enzyme• PstI – Providencia stuartii 164, 1st enzyme• Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme• KpnI – Klebsiella pneumoniae, 1st enzyme

Restriction Endonucleases

• Enzymes that cut at same position on both strands leave “blunt” ends SmaI

5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’

Enzymes with staggered cuts complementary ends

• HindIII - leaves 5´ overhangs (“sticky”)

5’ --AAGCTT-- 3’ 5’ --A AGCTT--3’

3’ --TTCGAA-- 5’ 3’ –TTCGA A--5’

• KpnI leaves 3´ overhangs (“sticky”)

5’--GGTACC-- 3’ 5’ –GGTAC C-- 3’

3’--CCATGG-- 5’ 3’ –C CATGG-- 5’

Actions of restriction enzymes-overview

Recombinant techniques

• DNA to be cloned and the vector molecule are  treated with the same restriction nuclease separately

• It produces complimentary sticky ends• Sticky ends will self ligate through covalent bonding

• This results in recombinant DNA molecule

Ligation of DNA

DNA Ligases close nicks in the phosphodiester backbone of DNA

DNA ligase is a enzyme that can link together DNA strands that have double-strand breaks (a break in both complementary strands of DNA).

Needs ATP

ATP

Cloning-Transformation

• It is introduced into host cell by adding it into culture of plasmid free bacteria or animal cells. 

• Heating and adding calcium chloride favors the transformation

• Once inside the host cell, the recombinant DNA begins to multiply and form the desired product.

Selection of recombinant cells

Selection of recombinant cells

• Only bacteria which have taken up plasmid grow on ampicillin.

• Blue-white selection:– white colonies have

insert– blue colonies have no

insert

Growing successfully….

• The transformed cell are cultured and multiplied.• Colony of cell each containing the copy of the recombinant plasmid is obtained.

Non-Bacterial transformation

Microinjection, using micropipette.

The host cells are bombarded with high velocity micro-projectiles, such as particles of gold or tungsten that have been coated with DNA.

Phage Introduction

• Phage is used instead of bacteria.• In vitro packaging of a vector is used.• lambda or MI3 phages to produce phage

plaques which contain recombinants.

Electroporation

• It involves applying a brief (milliseconds) pulse high voltage electricity to create tiny holes in the bacterial cell wall that allows DNA to enter.

Applications… Pharmaceutical and Therapeutic Applications  

        Gene therapy Medical diagnosis Xenotransplants Agricultural Applications

      Production of transgenic organisms

Environmental applications

• Many waste products of agriculture/industry do not break down naturally/break down slowly. 

Many bacteria have been GE capable of breaking down oil and other organic wastes in Cheese making industry : GE Saccharomyces cerevisiae able to dispose of whey by converting lactose to alcohol.

 Agricultural waste products, eg. corn husks, contain cellulose that normally decomposes slowly, can be converted into sugar by cellulase. Cellulase has been inserted in E.coli making it useful in waste management/disposal programs..

Any Questions??????...