Recombinant DNA technology Production of Recombinant DNA DNA

Recombinant DNA technology

Production of Recombinant DNA (DNA cloning principle) First steps Cutting DNA of interest by restriction enzyme, then insertion of this DNA into plasmid vector to produce recombinant DNA Second steps Replication of recombinant DNA (chimeric or hyperid) to produce large amount of new DNA (cloning) By the same restriction enzyme cut the DNA after replication , this new DNA can used in TTT of diabetes mellitus

Restriction enzyme (endonuclease) Restriction enzyme are bacterial enzyme that recognize and act in short part of DNA and cut it at specific sequence range from 4 -6 bp this sequence is palindromic Palindrome is a sequence that read from 5 to 3 in top as 5 to 3 in bottom 5 -GAATTC – 3 3 -CTTAAG-5 PALINDROME

Type of restriction enzyme cut axis of symmetry Blunt end This enzyme make cut at the axis of symmetry 5 -GAA TTC – 3 3 -CTT AAG-5

Sticky end This enzyme make cut that the 2 end overlapping each other 5 -G AATTC – 3 3 -CTTAA G-5

Restriction enzyme don’t make cut to itself in bacteria because it Protect itself by methylation on base

DNA amplification 1 - Types of cloning vectors (in vivo) Plasmid They are small circular double DNA Their function is to give antibiotic resistance to bacteria They carry from 5 -10 kilo base Replicate independent on bacterial cell All plasmid sequence are known so easy location of restriction enzyme Separate easy from bacterial DNA

Phages They are type of virus live in bacteria They have linear DNA They carry from 15 -20 kilo base Cosmid Plasmid act as a vector for large fragment They carry from 35 -50 kilo base

Bacterial artificial chromosome (BAC) They carry from 50 - 250 kilo base Yeast artificial chromosome (YAC) They carry from 500 - 3000 kilo base Importance of DNA cloning 1 -Vaccine synthesis 2 - Insulin synthesis 3 - Diagnosis of some disease

Application of recombinant DNA technology Diagnostic function Diagnostic of some disease as sickle cell anemia Production of vaccine for hepatites B Used in forensic medicine to diagnose blood sample Predict risk of developing disease Diagnosis of neonatal genetic disease

Therapeutic function TTT of sickle cell anemia by gene therapy Production of insulin in TTT of diabetes TTT of thalasemia by gene therapy

2 -Polymerase chain reaction (PCR) in vitro Definition : its technique used to amplify DNA or RNA up to million fold Technique (thermal cycler) Denaturation steps Double stranded DNA heated to separate into single strand Annealing steps RNA primer added to allow synthesis of new strands Then cooling to form new DNA Extension steps To make the cycle repeated

Importance of PCR 1 -Used for detection of viruses as hepatites C 2 -Diagnosis of neonatal genetic disease 3 -Used in forensic medicine 4 - Used in tissues typing in transplantation 5 - Used in archeological sample Advantages 1 - Sensitive 2 - Faster 3 - Less difficult

Libraries 1 -Genomic library Its collection of cloned fragment that represent the whole genome (both intron and exon) prepared by partial digestion of total DNA with restriction enzyme

2 - Complementary DNA (c. DNA) library: its collection of cloned fragment that represent the exon (m. RNA)only prepared by expression vector DNA transcription m. RNA c. DNA reverse transcriptase (Rt) m. RNA

Genomic library Complementary. 1 DNA (c. DNA) Its collection of. 1 its collection of cloned. 1 cloned fragment that represent the whole the exon only genome (both intron and exon) 1. prepared by partial Carried on expression. 2 digestion of total DNA vector with restriction enzyme 2. Prepared from DNA 3. Prepared from RNA by reverse transcriptase

Probes Pieces of DNA or RNA labeled with radioactive P 32 Types 1 - Radioactive with P 32 2 - Non radioactive use chemical or coloured substance Analysis of DNA and RNA Southern technique detect DNA Northern technique detect RNA western technique detect protein

Southern technique for DNA Cleavage of DNA by restriction enzyme Electrophoresis of DNA is done by agarose gel electrophoresis Blotting : transfer of DNA from agarose gel to nitrocellulose paper Autoradiography Detection of radioactive DNA by photographic film

Northern blot (RNA) 1 - Cleavage of RNA by restriction enzyme 2 -Electrophoresis of RNA is done by agarose gel electrophoresis 3 -Blotting : transfer of RNA from agarose gel to nitrocellulose paper 4 -Autoradiography Detection of radioactive RNA by photographic film

Western blot (for protein) Make electrophoresis Detection of protein by specific antibody

Gene mapping It s localization of specific gene on chromosome Gene mapping give information about human disease

Gene therapy Aim : to replace defective gene by a new one Mechanism Ex vivo gene delivery: Take cell from patient then put the gene and return it to patient In vivo gene delivery: Put gene directly into cell of patient

DNA polymorphism (marker) Its variation in DNA sequence from one person to another Restriction fragment length polymorphism (RFLP) Cutting of DNA by restriction enzyme produce many different fragment Identification of these fragment by making DNA analysis (Southern technique) Function of RFLP Diagnosis of sickle cell anemia Cystic fibrosis of pancreas Chorea which is neurological disease

2 - Variable number of tandem repeat Microsatellite polymorphism Has tandem repeat of AC sequence on DNA The number of nucleotide repeated is from 1 6 Used in genetic engineering Minisatellite polymorphism Has tandem repeat of sequence on DNA from 10 -100 Used as DNA finger print and determine paternity

3 - Single nucleotide polymorphism Result from base substitution caused by mutation or radiation It occur in one base every 1000 -3000 bp in human