Recombinant dna technology- a perforated insightSUBMITTED BY:VISHNU.R.NAIRTHIRD YEAR PHARM.DSUBJECT: PHARMACOLOGYCOLLEGE: NATIONAL COLLEGE OF PHARMACY (NCP)KERALA UNIVERSITY OF HEALTH SCIENCES (KUHS), KERALA STATE.

INDEX:• GENERAL PROPERTIES

• HISTORY OF RECOMBINANT DNA TECHNOLOGY

• PRINCIPLE INVOLVED

• MOLECULAR TOOLS OF GENETIC ENGINEERING

• HOST CELLS USED FOR RECOMBINANT DNA TECHNOLOGY

• VECTORS (VEHICLES) USED

• METHODS OF GENE TRANSFER

• GENE CLONING STRATEGIES

• APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY

• BIBLIOGRAPHY

GENERAL PROPERTIES :• Refers to “ A series of procedures, used to RECOMBINE DNA SEGMENTS”

• Under certain conditions a recombinant DNA molecule can enter a cell replicate

• Recombinant DNA can be defined as:

“ DNA, that has been engineered, by SLICING TOGETHER DNA FRAGMENTS from MULTIPLE SPECIES and INTRODUCED into the CELLS of a HOST”………………………

History :• One of the recent advances in BIOTECHNOLOGY

• Developed by 2 scientists, namely BOYER and COHEN , in 1973…………………

PRINCIPLE INVOLVED :• Principle of RECOMBINANT DNA TECHNOLOGY can be explained in the form of the

following flowchart:

GENERATION of DNA fragments , and SELECTION of desired DNA piece (eg : human gene)

Insertion of selected DNA into a CLONING VECTOR ( Plasmid)

RECOMBINANT DNA formed (CHIMERIC DNA)

CONTINUED……………………………..

Recombinant vectors introduced into host cells (BACTERIA)

Multiplication and selection of clones containing recombinant molecules

GENE EXPRESSION done

Desired Product is obtained………………….

Molecular tools of genetic engineering :

• Here , we are discussing about the enzymes that are used in Recombinant DNA Technology.

• Enzymes include:

1. RESTRICTION ENDONUCLEASES:

- Also known as “DNA cutting enzymes”

- Important group of enzymes for DNA manipulation

- Bacterial enzymes, that can cut/split DNA at specific sites

- Discovered in E.Coli, that restricted the replication of bacteriophages (Hence the name RESTRICTION ENDONUCLEASE or RESTRICTION ENZYMES)

CONTINUED………………………………..

• Examples of Restriction endonucleases include:

a. ECoRI (From E.Coli)

b. Hind III (From Haemophilus influenzae)

c. Hae III (From Haemophilus aegyptius)

d. Bam HI ( From Bacillus amyloliquefaciens)

e. NotI (From Nocardia otitidis)……………………..

CONTINUED………………………………..

2. DNA LIGASES:

- Cut DNA fragments Joined covalently by DNA ligases

- Originally isolated from viruses

- Also occur in E.Coli and eukaryotic cells

- Actively participate in cellular DNA repair process

- Permanently hold DNA pieces together………………………

CONTINUED……………………………………..

3. MISCELLANEOUS ENZYMES:

a. ALKALINE PHOSPHATASE:

Removes phosphate groups from 5’ ends of single stranded or double stranded DNA/RNA

b. Bal 31 NUCLEASE:

For progressive DNA shortening

c. REVERSE TRANSCRIPTASE:

Synthesizes DNA from RNA

CONTINUED…………………………………

d. SI NUCLEASE:

Degrades single stranded DNA/ RNA

e. Taq DNA Polymerase :

Used in PCR (Polymerase Chain Reaction),etc……………………….

Host cells used for recombinant dna technology :• Microbes multiply faster compared to cells of higher organisms (plants/ animals) Thus preferred

as host cells

• Prokaryotic Hosts include:

- E.Coli

- Bacillus subtilis

• Eukaryotic Hosts include:

- Saccharomyces cerevisiae (Fungi)

- Mammalian cells ( Mouse cells)

- Plants (Intact cells, protoplasts etc)………………………..

VEHICLES / VECTORS USED :

• Vectors are DNA molecules, that carry a foreign DNA segment (fragment) to be cloned

• Self-replicating in an appropriate host cell

• Important vectors include:

a. PLASMIDS

b. BACTERIOPHAGES

c. COSMIDS

d. ARTIFICIAL CHROMOSOME VECTORS…………………..

CONTINUED………………………………

A. PLASMIDS:- Extrachromosomal

- Double stranded

- Circular

- Self-replicating DNA molecules

- Contribute 0.5-5 % of total bacterial DNA

- Examples:

• p BR 322 * p UC 19…………………..

CONTINUED……………………………………

B. BACTERIOPHAGES:- Also known as “phages”

- Viruses, that replicate within bacteria

- Phage vectors accept short fragments of foreign DNA into their genomes

- They can take up larger DNA segments than plasmids

- Preferred for working with genomes of human cells

- Examples:

* Bacteriophage lambda (phage lambda) * Phage M13………………………

CONTINUED……………………………………..

C. COSMIDS:- Possess characters of both plasmids and phages

- ‘Cos’ site of phage lambda DNA segment added to plasmids cosmids formed

- Foreign DNA inserted into cosmid DNA Recombinant DNA formed injected into E.Coli once inside host cell, cosmids behave like plasmids and replicate

- Carry larger fragments of foreign DNA compared to that of plasmids…………………………..

CONTINUED…………………………………………..

D. ARTIFICIAL CHROMOSOME VECTORS:

Are of 3 types:

i. Human Artificial Chromosome (HAC):

- Synthetically produced vector DNA

- Possesses features of human chromosome

- Also finds application in gene therapy

ii. Yeast Artificial Chromosome (YAC):

- Synthetic DNA

- Can accept large fragments of foreign DNA (Mainly human DNA)…………….

CONTINUED…………………………………….

iii. Bacterial Artificial Chromosome (BAC):

- Can accept large fragments of foreign DNA……………………..

Methods of gene transfer :

A. INTRODUCTION:- Introducing a foreign DNA (gene) into cell, is an important task in biotechnology

- Efficiency of this process is often crucial for determining cloning success

- Methods used include:

1. TRANSFORMATION

2. CONJUGATION

3. ELECTROPORATION

4. LIPOFECTION

5. DIRECT DNA TRANSFER…………………………….

CONTINUED………………………………………..

B. METHOD DETAILS:1. TRANSFORMATION:

- “Method of introducing foreign DNA into bacterial cells “ (example E.Coli)

- Uptake of plasmid DNA by E.Coli is carried out in ice-cold Calcium Chloride (0-5 degrees) subsequent heat treatment (37-45 degrees for about 90 seconds)

- By this technique, the transformation frequency can be made reasonable good (approx. 1 cell per 1000 cells)

- TRANSFORMATION FREQ. : “Fraction of cell population that can be transferred”……………………….

CONTINUED……………………………………………..

2. CONJUGATION:

- Natural, microbial recombination process

- During conjugation 2 live bacteria (1 donor and 1 recipient ) come together join by cytoplasmic bridges transfer of single stranded DNA from donor to recipient occurs

- Inside recipient cell New DNA may integrate within chromosome (rare case) / may remain free (as in plasmid)

- Mainly used for gene transfer

- This is achieved by transferring plasmid-insert DNA from one cell to another

- Only few plasmids possess conjugative properties to transfer DNA to recipient cells.........

CONTINUED…………………………………….

3. ELECTROPORATION:

- Principle involved:

High voltage electric pulses induce cell plasma membranes to fuse

- A technique, that involves ELECTRIC FIELD MEDIATED MEMBRANE PERMEABILIZATION

- Electric shocks form pores due to electric pulses cause induction of exogenous DNA from the suspending solution

- Simple and rapid technique for introducing genes into the cells from various organisms (microbes, plants and animals)…………………………..

CONTINUED…………………………………………….

4. LIPOSOME MEDIATED GENE TRANSFER:

- Also known as LIPOFECTION

- Liposomes circular lipid molecules possess an aqueous interior carries nucleic acids

- DNA fragment treated with liposomes these liposomes adhere to cell membranes fusion between liposomes and cell membranes occur DNA fragment transfer occurs Thus DNA enters into cell Later DNA gets integrated into the nucleus

- Positively charged liposomes Complex with DNA easily Bind to cell and transfer DNA rapidly……………………………………

CONTINUED………………

5. DIRECT TRANSFER OF DNA:

- It is possible to directly transfer DNA into nucleus

- Techniques used for this purpose include:

a. MICROINJECTION:

• Involves use of glass micropipette to inject a liquid at a microscopical/ borderline macroscopical level

• Mainly used for penetration into cell membrane and or nuclear envelope

CONTINUED…………………………

• Finds use in:

i. Cloning of organisms

ii. Study of cell biology and viruses

iii. To treat male subfertility through intra-cytoplasmic sperm injection (ICSI)

b. PARTICLE BOMBARDMENT :

- Usually comes under “gene gun”, “ bio ballistics”, “biolistics “ , or “ biolistic particle delivery system “

- Used for injecting cells with genetic information

CONTINUED……………………………….

• Can transform almost any type of cell, including plants

• Can transform genetic material of nucleus, and even organelles……………………………………………………

GENE CLONING STRATEGIES :• Clone is defined as:

“ Group of organisms, cells, molecules or any other objects , arising from a single individual”

• Gene cloning strategies in relation to Recombinant DNA Technology involves the following aspects:

1. GENERATION OF DNA FRAGMENTS:

- Methods used include Restriction endonuclease digestion, cDNA synthesis, PCR, Chemical synthesis

2. INSERTION INTO A CLONING VECTOR :

- Methods include Ligation of blunt/ cohesive ends, homopolymer tailing, linker molecules

CONTINUED…………………………………..

3. INTRODUCTION INTO HOST CELLS:

Involves Transformation, lipofection, etc………

4. SELECTION/ SCREENING:

Involves hybridization, PCR, immunochemical methods, protein-protein interactions, functional complementation………………………………………………..

APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY :

1. IN MOLECULAR BIOLOGY:- Used to ELUCIDATE MOLECULAR EVENTS in BIOLOGICAL PROCESSES like:

a. CELL DIFFERENTIATION

b. AGEING

c. GENE MAPPING, etc……………….

CONTINUED……………………………………..

2. IN MOLECULAR DIAGNOSIS OF DISEASES:- Used to check the genetic disorders in carrier parents, and their predictable chances of

producing afflicted children

- For identification of infectious diseases,like:

a. Food poisoning Salmonella

b. Pus forming Staphylococcus

c. Hepatitis virus

d. HIV………………………………..

CONTINUED………………………………………

3. APPLICATION IN MONOCLONAL ANTIBODIES:

-Monoclonal antibodies : “Specific lymphocytes , which after isolation and in culture vitro , produce a single type of antibody, with specificity against a single type of antigen”

- Such monoclonal antibodies find applications in the following:

a. Disease diagnosis:

• Streptococcal infections

• Chlamydial infections

CONTINUED……………………………

b. Diagnosis of pregnancy:

• Pregnancy diagnostic kit contains monoclonal antibodies of hormone excreted only in the urine of pregnant women

• Take urine sample of woman apply on the strip of diagnostic kit

• If woman is pregnant antigen-antibody reaction occurs color change is indicated

• If woman is not pregnant no reaction no color change occurs.

c. In treatment of cancer:

* In cancer treatment targeted drug delivery is driven by monoclonal antibodies thus, ONLY CANCEROUS CELLS RECEIVE CHEMOTHERAPY ,and the healthy cells are UNAFFECTED...........

CONTINUED………………………………………

4. APPLICATION IN GENE THERAPY:- GENE THERAPY is defined as “ Replacement of faulty / mutated genes with a healthy

gene”

- Research and studies are going on to find applications in diseases, ranging from SICKLE CELL ANEMIA to killer diseases like SCID…………………..

CONTINUED……………………………………

5. APPLICATION IN DNA FINGER PRINTING:- Every individual differs from other with respect to uniqueness in finger prints

- Thus, in order to obtain DNA fingerprints of an individual, it is important to look for genes that occur in multiple forms in different individuals……………………………

CONTINUED………………………….

6. IN VACCINE PRODUCTION:- Vaccines are defined as:

“Inactivated (killed) or attenuated pathogens, which when injected into the body of an organism, induces production of antibodies in the organism against the specific disease”

- Recombinant DNA Technology has been used for the commercial production of vaccines for diseases like:

a. Hepatitis B

b. Malaria

CONTINUED……………………………………..

c. Typhoid

d. Cystic cerosis caused by Taenia soleum

- Gene transfer mediated through Ti plasmid to plants produces edible vaccines …………………………………………

CONTINUED……………………………………..

7. APPLICATION IN COMMERCIAL AND PHARMACEUTICAL PRODUCTS:- Production of human insulin through Recombinant DNA Technology has revolutionized

diabetes management

- HUMAN INSULIN GENE CLONED INTRODUCED INTO E.COLI THIS GENETICALLY ENGINEERED E.COLI BACTERIA GROWN IN CULTURES PRODUCES HUMAN INSULIN

- There are miscellaneous other pharmaceutical products that are produced by Recombinant DNA Technology, that include the following :

CONTINUED………………………………

• PRODUCTION OF L-ASCORBIC ACID

• PRODUCTION OF ANTIBIOTICS…………………………………………………………

BIBLIOGRAPHY:• TEXTBOOK OF BIOCHEMISTRY BY U.SATYANARAYANA and U.CHAKRAPANI (Page

no. 576)

• An introduction to GENETIC ANALYSIS by GRIFFITHS, MILLER, LEONTIN, GELBART, 1996

• “RECOMBINANT DNA TECHNOLOGY: APPLICATIONS IN THE FIELD OF BIOTECHNOLOGY AND CRIME SCIENCES : by PANDEY SHIVANAND, SUBA NOOPUR

• www. Preserve articles. Com

• www.wikilectures.eu.index conjugation,transformation

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