•Recombinant DNA technology

•Biotechnology •And

• Recombinant DNA (rDNA)

•**Since World War I microbes have been used to produce a variety of chemicals=== (ethanol, acetone, citric acid …)

•**Since World War II microbes used to produce certain antimicrobial agents=== antibiotics

•**Now microbes are being used as factories to produce chemicals that the organism do not naturally make

•Biotechnology

•Biotechnology == is the use of m.o, cell or cell component to make a product

• by•inserting genes into cells by rDNA technology ==

genetic engineering

•**Gene from vertebrate animal (man) can be inserted into the DNA of a bacterium

•**the recipient can then be made to express the gene, which may code for a commercially useful product

•**Human insulin ------ treating diabetes

•**Vaccine for hepatitis B virus

•**Human growth hormone

•**Make thousands of copies of the same DNA molecule to amplify DNA ----experiments & analysis for identifying microbes (viruses)

Bacterial genetic material

Bacterial chromosomeSingle,long,D.S DNA = contain genes carry all genetic information required for bacterial structure and function

Plasmids=5-100 genes not needed for growth & reproduction == (survival in stressful situation)Small,circular,D.S. DNAExtra chromosomal genetic material

replicate independently of chromosomal DNA

DNA structure•DNA molecule is composed of

repeating units of nucleotides===are composed of deoxyribose sugar molecule linked to a phosphate group and to a nitrogenous base (adinine-thymidine, cytosine-guanine)

•Nucleotides are twisted together in pairs to from a double helix ---the 2 strands are held together by hydrogen bonds

•The 2 strands are complementary

•Duplication of DNA during division

•==replication

•DNA polymerase

•Genetic code•Set of rules that determines how a

nucleotide sequence is converted into the a.a. sequence of a protein that control cell activities

•Transcription==synthesis of a complementary strand of RNA(carry all information) from DNA template by RNA polymerase

•Translation==protein synthesis==is the process in which the information in the nucleotide base sequence of mRNA is used to dictate the a.a. sequence of a protein (in 3 base segments)

•---this associated with ribosome which consist of rRNA and protein

•Genetic transfer & Recombination

Genetic transfer & Recombination•Genetic recombination==exchange of genes between

two DNA molecules to form new combinations of genes on

a chromosome by crossing over

• the chromosome break & then rejoin

•The original chromosomes carry a portion of the others gene• (carry a new function)

Recombination/ Crossing over

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rejoin

Chromosomes== different than either parents

Genetic transfer•Involves a

•donor cell (give portion of its DNA)• and• a recipient cell

•Part of donors DNA is usually incorporated into the recipient DNA

•The recipient === recombinant

How can gene transfer from one bacterial cell to other?

recombination

Transformation

How can gene transfer from one bacterial cell to other?

•Transformation===genes transferred as naked DNA in solution==occurs naturally

•***after bacterial death or lyses•***some bacteria release their DNA into the environment***other bacteria of the same species can come in contact with

these fragments

***take them up and incorporate them into their DNA by recombination

• #### Recombinant cell ###•Has different genetic makeup than the donor or the recipient

Transduction

How can gene transfer from one bacterial cell to other?

•Transduction=== involvebacteriophage==virus that infect bacteria

•***transfer DNA from one bacterium •to another

•

How can gene transfer from one bacterial cell to other?

•Conjugation==require contact between living cells and the presence of a special plasmid

•F plasmid ==facilitates conjugation•== can give a bac. new genes that may

• help it survive in a changing• environment

•==25 genes that code for production of pilli

•F+ == have F plasmid

•F- == have no F plasmid

•This event occurs when bacteria extend sex pilli to attached the cytoplasmic bridge and F plasmid transferred to the recipient cell and integrated within the chromosome (crossing over)

•Recombinant genome can be passed on to future generations

Other plasmid of equal importance•R plasmid== resistance plasmid =transferred by

conjugation and pass to future generation• == carry genes that confer

• resistance to certain antibiotics•

•Bacteria with R plasmid•

•Bacteria without R plasmid•

code for enzymes that inactivate certain drugs

make the drug inactive

sensitive to that antibiotic and destroyed by it

Recombinant DNA technologies

• ###A desired gene is inserted into a DNA vector (plasmid or viral genome)

• ###The vector inserts the DNA into a new cell which is grown to form a clone

• ###Large quantities of the gene product can be harvested from the clone (protein)

Molecular cloning•**is a set of experimental methods in molecular biology that

are used to assemble recombinant DNA molecules and to direct their replication within host organisms

•Cloning== replication of a single DNA molecule starting from a single living cell to generate a large population of cells

containing identical DNA molecules .•Molecular cloning generally uses DNA sequences from two

different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA.

Molecular cloning

Molecular cloning

•Cloning== replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing

identical DNA molecules .•Molecular cloning generally uses

DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA.

•**is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms

Molecular cloning experimentDNA to be cloned is obtained from an organism of interest

Tools of rDNA technology

•Microbes & vectors•== Lab.strain of bacteria (E.coli)

•== plasmid cloning vector

•**widely available•**versatile

•**offer rapid growth•**smaller size are more easily manipulated

•**circular (destruction)

Tools of rDNA technology

•Enzymes•** restriction enzyme

•==recognizes & cuts only one particular nucleotide sequence in DNA-----some produce sticky ends of the DNA fragments

•** DNA ligase•==covalently link the DNA back bones by base pairing---

rejoin DNA pieces

•Endonuclease•Alkaline phosphatase•Reverse transcriptase

•

Tools of rDNA technology

•DNA to be cloned•== extracted from the organism of

interest*any tissue source can be used (extinct animals)

•*DNA also can be obtained from RNA using reverse transcriptase

•== The extracted DNA is then purified to remove contaminating proteins

•==Amplified

Method

DND ligase

cloning

Application of rDNA technology• provides an essentially unlimited quantity of any

DNA segments derived from any genome. This material can be used for a wide range of purposes, including those in both basic and applied biological science.

•recombinant protein ===Obtaining the molecular clone of a gene can lead to the development of organisms that produce the protein product of the

cloned genes.

•1-medically useful proteins ==whose administration can correct a defective or poorly expressed gene

•** factor VIII a blood-clotting factor deficient in• some forms of hemophilia

•** insulin used to treat some forms of diabetes

•2-proteins ==that can be administered to assist in a life threatening emergency

• **tissue plasminogen activator used to treat strokes

• 3-recombinant subunit vaccines == purified protein can be used to immunize patients against infectious diseases, without exposing them to the infectious agent itself

•** hepatitis B vaccine •4 -recombinant proteins == standard material

• ** diagnostic laboratory tests

•Other application•Probes= DNA with known genetic sequence

•** nucleic acid hybridization•** antibody probes

•** polymerase chain reaction (PCR)•** DNA sequencing

•Transgenic organisms==genetically modified organisms (GMO)

•** transgenic mouse•** commercial use (pharmaceu.,plants,

• pharming)

Gene therapy

• ***supplying a functional gene to cells lacking that function----with the aim of correcting a genetic disorder

•fix a faulty gene by replacing it with a healthy gene

• ===the use of DNA as a drug to treat disease by delivering therapeutic DNA into a patient's cells

SCID=Sever Combined Immunodeficiency Diseasedue to a defective gene for Adenosine Deaminase (ADA)

First Term Exam.

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