Recombinant Dna

Genetic engineerin g? No idea. But you can ask me about aliens…

RECOMBINANT DNA TECHNOLOGY/ GENETIC ENGINEERING By: Feliza Maren Israelita R. Barican

Review on Genetics

Deoxyrubonucleic Acid (DNA) • Double stranded – Composed of a sugar (deoxyribose), a phospate residue and one of the four bases: adenine (A), cytosine (C), guanine (G) and thymine (T) • Base Pairing ; Purine – Pyrimidine • DNA Replication

Ribonucleic Acid • Single stranded – Consist of a ribose sugar, a phospate residue and one of the four bases: adenine (A), cytosine (C), guanine (G) and uracil (U) • RNA species; rRNA, tRNA, mRNA • Role of mRNA; intermediate product b/w amino acid and DNA • Hybridzation – strands of nucleic acids binding together forming

Replication

dsDNA ssDNA

Transcription dsDNA

AMINO ACID

mRNA

Reverse Transcription

dsDNA ssDNA

Translation mRNA

PROTEIN

Biotechnology

• The industrial application of microorganisms, cells or cell components to make a useful product.

Recombinant DNA Technology • Manufacturing and manipulating genetic material in vitro. • Its product is called recombinant DNA.

Recombinant/ Chimeric DNA • Synthetic DNA that is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences that would not normally occur together.

Chimeric Molecules • Recombinant DNA that is further changed to host additional strands of DNA. • Regular in occurrence • Propagation by vectors ensures the presence of hundreds of thousands of organismal and bacterial cells that all contain copies of the original chimeric

Recombinant DNA Technology Procedures

DNA Isolation • Cells in a broth culture are lysed by using a detergent to isolate bacterial DNA. • Burst cells release fragile DNA. • The fragile DNAs are treated with specific enzymes to purify the obtained DNA by removing unnecessary RNA and proteins.

Examples of Enzymes Used in Recombinant DNA Research

ENZYME

PRIMARY ACTION

PRIMARY USE

Alkaline phosphatase

Dephosphorylates 5’ ends of RNA and DNA

BAL 31 nuclease

Degrades both the 3’ and 5’ ends of DNA Catalyzes bonds between DNA molecules Synthesize dsDNA from ss DNA

Removal of 5’ PO4 groups prior to kinase labeling to prevent self ligation Progressive shortening of DNA molecules Joining of DNA molecules

DNA ligase DNA polymerase I

DNAse I

Synthesize dsDNA; Nick translation; generation of blunt ends from sticky Under appropriate Nick translation; ends conditions, mapping of produces ss nicks in hypersensitive DNA sites; mapping protein DNA interaction

Exonuclease III λ exonuclease Polynucleotide kinase Reverse transcriptase SI nuclease

Terminal transferase

Remove nucleotide from 3’ ends of DNA Removes nucleotides from 5’ ends of DNA Transfer terminal phospate (γ position) from ATP to 5’ OH groups of Synthesize DNA or RNADNA from RNA template

DNA sequencing; mapping of DNA protein interactions DNA sequencing 32P labeling of DNA and RNA

Synthesis of DNA from mRNA; RNA c5’ end mapping study Degardes single Removal of hairpin stranded DNA in synthesis of cDNA; RNA mapping Adds nucleotides to Homopolymer the 3’ ends mapping

Using Restriction Enzymes to Generate DNA Fragments • Purified DNA is cut (or “digested”) into smaller fragments by restriction enzymes. • Digestion of DNA with restriction enzymes generates blunt-ended DNA or sticky ended (or

Restriction Enzymes • An enzyme that causes cleavage of both strands of DNA at highly specific sites dictated by the base sequence. • First isolated in 1970, when certain bacteriophages were found to have a restricted host range. • Protects a bacterial cell by hydrolyzing phage DNA.

• Named after the bacterium in which they are isolated. • Recognizes palindrome (repeated, inverted) gene sequence

Examples of Restriction Enzymes

RESTRICTION ENDONUCLEA SE Bam HI

SEQUENCE RECOGNIZED CLEAVAGE SITES GGATCC SHOWN

BACTERIAL SOURCE Bacillus amyloliquefaciens H Bacillus glolbigii

Bg III

AGATCT

Eco RI

GAAATTC

Escheriecia coli RY13

Eco RII

CCTGG

Escheriecia coli R245

Hind III

AAGCTT

Haemophilus influenzae Rd

GCGC

Haemophilus haemolyticus

Hha I

Hpa I

GTTAAC

Haemolyticus parainfluenzae

Mst II

CCTNAGG

Microcoleus strain

Not I

GCGGCCGC

Nocardia otitidis caviarum

Pst I

CTACAG

Providencia stuartii

Sma I

CCCGGG

Serratia marcescens

Taq I

TCGA

Thermus aquaticus YTI

Types of DNA Fragments

Blunt-Ended DNA

• Two strands of DNA duplex having ends that are flush with each other. • Example; Alu I

Sticky (Cohesive) Ended DNA • Complementary single strands of DNA that protude from opposite ends of a DNA duplex or from the ends of different duplex molecule – Example; Bam HI

Using DNA Ligase to Join Vector and Insert • VECTOR – Plasmid or bacteriophage into which foreign DNA can be introduced for the purpose of cloning • INSERT – Additional length of a base pairs in DNA

Properties of an Ideal Vector • Replicator (“ori”) • Selective markers resistant to antibiotics • Multiple cloning sites

lacZ gene

CLEAVA GE SITES

AMP R

ORI

Examples of Cloning Vectors

Bacteriophage

Virally infected bacterium

Cosmid

A plasmid into which the DNA sequence from bacteriophage lambda that are necessary for packaging of DNA Small, extrachromosomal, circular molecule of DNA that replicates indepently of host DNA; with cos sites Derivatives of the F plasmid of E. coli; contains inserts as large as 300,000 nucleotieds in length

Plasmid

Bacterial Artificial Chromosome (BAC)

Yeast Artificial Chromosome Stores 1,000,000 nucleotides in (YAC) length Expression Vector Engineered to certain and appropriate protomer and ribosmome binding site adjacent to multiple cloning site Ti Plasmid Tumor inducing plasmid (Agrobacterium tumefaciens

• BACTERIOPHAGE T4 DNA LIGASE – Glues the DNA fragments together. – Forms covalent bonds between sugar phosphate residue of adjacent nucleotide .

Introducing Recombinant DNA into a New Host • Selecting a suitable host • Introducing DNA into cells • Selecting for transformants

Selecting a Suitable Host • Escheriecia coli – Most preferred host • Easily grow in laboratory • Can’t grow in normal environmental conditions found outside the laboratory

Introducing DNA into Cells

• DNA MEDIATED TRANSFORMATION – Cells take up DNA from surrounding environment.

• ELECTROPORATION – Inducing an electrical current to form microscopic pores in the membrane of the cells; DNA enters the cells through the cells.

• MICROINJECTION • DNA introduced directly into an animal cell.

Genetic Cloning • GENETIC LIBRARIES – Collection of genetic clones containing different DNA fragments. • Reverse transcriptase –An enzyme that is produced by artificial genes.

Selecting for Transformants/Clones • Blue white screening • Colony hybridization

Blue-White Screening • Color of bacterial colonies formed at the end of the screening process – (+) – Blue – ( - ) – White

Colony Hybridization • Identification of a colony containing a desired gene by using a DNA probe that is complementary to the gene.

Applications of Recombinant DNA Technology

Production of Recombinant Vaccines • RECOMBINANT VACCINE – Vaccines produced by recombinant DNA technology – Contains either a protein or a gene encoding a protein of a pathogen origin that is immunogenic and critical to the pathogen function

Pharmaceutical Products of Genetic Engineering

PRODUCT Alpha-interferon Antitrypsin

COMMENTS Therapy for leukemia, melanoma and hepatitis, prduced by E. coli and S. cerevisiae (baker’s patients; yeast) Assist emphysema produced by genetically modified sheep

Beta-interferon

Treatment for multiple sclerosis; produced by mammalian cell culture

Bone morphogenic proteins

Induces new bone formation; useful in healing fractures and reconstructive surgery; produced mammalian cell culture Counteracts effects of chemotherapy;

Colonystimulating factor improves resistance to infectious (CSF) disease such as AIDS; treatment of leukemia; produced by E. coli and S. cerevisiae (baker’s yeast)

Epidermal growth factor (EGF) Erythropoietin (EPO) Factor VII Gammainterferon HBV Human Growth Hormone (hGH)

Heals wounds, burns, ulcers; produced by E. coli Treatment of anemia; produced by mammalian cell culture Treatment of hemophilia; improves clotting; produced by mammalian cell culture Treatment of CGD; produced by E. coli Produced by S. cerevisae that carries hepatitis virus gene on a plasmid Corrects growth deficiencies in children; produced by E. coli

Influenza vaccine

Trial vaccine made from E. coli or S. cerevisiae carrying virus genes

Interleukins

Regulates the immune system; possible treatment for cancer; produced by E. coli Possible therapy for cancer and transplant rejection; used in diagnostic tests; produced by mammalian cell culture (from fusion of cancer cell and antibody-producing Monoclonal antibody used in cell) transplant patients to help suppress

Monoclonal antibodies

Orthoclone

Prourokinase Human insulin

the immune system, reducing th echance of tissue rejection; produced Anticoagulant; theray for heart by mouse cells attacks; produced by E. coli and yeast Therapy for IDDM; better tolerated than insulin extracted from animals; produced by E. coli

Pulmozyme (rhDNAse) Relaxin Superoxide dismutase

Taxol

Enzyme used to breakdown mucuos secretion in cystic fibrosis patients; produced by mammalian cell Usedculture to ease childbirth; produced by E.coli Minimizes damage caused by oxygen-free radicals when blood is resupplied to oxygen deprived tissues; produced by S. cerevisiae and pastoris PlantPichia produced used for treatment of ovarian cancer in E. coli

Tissue plasminogen activator (Activase) Tumor necrosis factor (TNF)

Dissolves the fibrin of clots; therapy for heart attacks; produced by mammalian cell culture Causes disintegration of tumor cells; produced by E.coli

Other Applications of Recombinant DNA Technology

• Normal Gene Variation – Human Genome Project • Disease-Causing Gene Variation • Xenotransplant • Nucleotide Sequence

“We cannot help ourselves from growing wiser. Those who are helpless to their search for knowledge are those who can’t transcend

Thanks for listening! Have a nice day!