RECOMBINANT DNA TECHNOLOGY

Dr. Balram SaiPrasanna Postgraduate in Pharmacology CAIMS

Basic structure of DNA

Basic structure of DNA

Recombinant DNA

rDNA is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together through the process of gene splicing

In a nutshell

DNA + plasmid (vector) recombinant vector transfected into bacteria spread plate of transformed bacteria colony selected and cloned DNA libraries application

Enzymes

Restriction enzymes

Cut DNA at specific sites Isolated from bacteria restricted the growth of bacteriophages Accompanied by site-specific DNA methylases restriction modification system Named after the bacterium from which they are isolated Cuts result in blunt ends or sticky ends

Restriction Enzymes

Restriction endonucleases

Categorised into four general groups based on

Their composition Enzyme cofactor requirements Nature of their target sequence Position of their DNA cleavage site relative to the target sequence

Restriction endonucleases

Type I enzymes :

Cleave at sites remote from recognition site Require both ATP and S-adenosyl-L-methionine to function Multifunctional protein with both restriction and methylase activities Cleave within or at short specific distances from recognition site Require magnesium Independent of methylase

Type II enzymes :

Restriction endonucleases

Type III enzymes :

Cleave at sites a short distance from recognition site Require ATP (but doesn't hydrolyse it) S-adenosyl-L-methionine stimulates reaction but is not required Exist as part of a complex with a modification methylase Target methylated DNA

Type IV enzymes :

Artificial restriction enzymes

Can be generated by fusing a natural or engineered DNA binding domain to a nuclease domain Such artificial restriction enzymes can target large DNA sites (up to 36 bp) and can be engineered to bind to desired DNA sequences E.g. : Zinc finger nucleases

DNA Recombination

Limitations of sticky-end ligation

Sticky ends of a vector may reconnect with themselves Fragments can also anneal back Sticky-end sites may not be available in a convenient position

Alternatives?

Enzyme that generate blunt end is used New ends are added using terminal transferase Homopolymer tailing Ligated using DNA ligase

Vectors

Plasmids Phages Cosmids Bacterial artificial chromosome (BAC) Yeast artificial chromosome (YAC)

Inserting rDNA into host

Adding calcium chloride makes bacterial cell membrane permeable to plasmids When vector has aspects of a virus, host cell can be transfected with recombinant molecule Electroporation Lipofection

Selecting colonies

DNA Libraries

Genomic library

Contain total DNA of a cell line or tissue Comprises cDNA corresponding to the gene expressed in the tissue

cDNA library

DNA Libraries
Genomic Library Source of DNA Enzymes Chromosome Restriction endonuclease, DNA ligase Yes Not necessarily Yes Yes cDNA Library mRNA Reverse transcriptase, DNA ligase No Yes No No

Non-expressed sequences Complete sequences Introns Promoter and enhancer sequences

DNA Libraries - Uses

Genomic libraries used to identify

Protein coding genes Restriction endonuclease sites Genetic markers (STR, SNP) Non-expressed DNA

DNA Libraries - Uses

cDNA libraries used to

Sequence specific genes Identify disease-causing mutation Produce recombinant proteins (expression vectors) Gene therapy Produce transgenic animals

Practical applications of recombinant DNA technology

Production of recombinant proteins Advantages:

Production in large amount compared to conventional methods Provide human material

Recombinant proteins

Human recombinants that largely replaced animal or harvested from human types

Human growth hormone Human insulin Follicle-stimulating hormone Factor VIII

Recombinant proteins

Human recombinants with recombination as only source

Erythropoietin G-CSF apha-galactosidase A alpha-L-iduronidase N-acetylgalactosamine-4-sulfatase Dornase alfa Tissue plasminogen activator Glucocerebrosidase Interferons Insulin-like growth factors

Recombinant proteins

Animal recombinants

Bovine somatotropin Porcine somatotropin Bovine chymosin Envelope protein of hepatitis B virus (vaccine)

Viral recombinants

Uses

Used in the molecular analysis of diseases

Normal gene variation Gene variations causing diseases Point mutations Deletions, insertions and rearrangements of DNA Pedigree analysis Prenatal diagnosis Restriction fragment length polymorphisms (RFLP) and single nucleotide polymorphisms (SNP)

Uses (contd..)

Microsatellite DNA polymorphisms RFLPs and variable numbers of tandemly repeated units (VNTRs) in forensic medicine Gene therapy Transgenic animals Targeted gene disruption (knockout) RNA transcript and protein profiling

High-density microarray technology Transcriptome information Mass spectrometry of complex protein samples

Bibliography

Harper's Illustrated Biochemistry Jeremy M Berg - Biochemistry Text Book of Biochemistry Devlin Kaplan, Colowick, Wu Recombinant DNA Cohen SN et. al. - Construction of biologically functional bacterial plasmids in vitro Roberts RJ et. al. - Restriction endonucleases Pingoud, Alves, Geiger Enzymes of Molecular Biology