Biotechnology
Mendle work => rediscovered in 1900 => geneticists => lead to new insulin => gene removed from one organism and inserted era of technology DNA into another to obtain a desired substance.
Gene Therapy => to repair a fault gene.
clonning => many identical copies and exact copies. A s€xual reproduction. identical to original specimen.
Recombinant DNA Technology: Known as genetic regenering. Produces large quantity of genes as compared to PCR. Uses enzyme to cut and paste together DNA sequence of interest.
DNA from two different sources:
Following are required for recombinant DNA Technology.
1. Gene of interest => which is to be clonined.
2. Molecular scissors => to cut gene of interest.
3. Molecular vector/ carne => on which gene of interest placed.
4. Gene of interest along with vector => introduced into expression system => specific product made.
Three way to isolate gene of interest:
• To isolate from chromosome => by culting chromosome of flanking.
• To synthetic chemically.
• To make if from mRNA. Sites of gene => Special enzyme restriction endonuclease.
mRNA => reverse transcriptase => DNA => cDNA => DNA from reverse transription.
Restriction Endonuclease:
molecular Scissors => 1970 => Hamilton O Smith => first Restrictions enzyme. Natural enzymes of bacteria => which they used against protection from virus. Restrict the growth of virus => Restriction endonuclease.
Cut DNA at specific sites =>Specific sequence of 4 – 6 nucleotides arranged in reverse order. 400 enzymes isolated => 20 used in recombinant DNA technology.
e.g: ECOR1 => cut double standard used in crisper.
DNA at cleavage site => gap is formed => on which foreign DNA placed end bases complementary to those exposed by restrictions enzyme. Single stranded complementary ends of two DNA molecule sticky ends => binds to complementary bas pairing.
Molecular carrion / vector:
Recombinant select vector =>means by which recombinant DNA introduced into host cell.
e.g: Type => plasmid => in E-coli.
Plasmid => natural extra => chromosomal circular DNA having gene for antibiotic resistance and fertility. PSC101 => resistant gene for tetracyclin. PBR322 => antibiotics gene for tetracyclin and ampicillin.
Recombinant DNA:
Plasmid cut => same enzyme => used in gene of interest isolation. Gene of interest (Insulin) => joined with sticky ends => DNA ligase => joins foreign DNA into vector => Two different pieces of DNA joined foreign => Recombinant DNA or chimeric DNA.
Bacterial cell => take recombinant plasmid => when treated with CuCl² => make then permiable. Bacterial cell => each new cell contain attract one plasmid => each bacteria gene of interest => express => make new products.
Besides plasmid => DNA of bacterial viruses (lambda phase) can be used as vector.
Genomic Library:
Full set of gene in individual. Collection of bacterial clone or bacterial phase => each clone contain particular segment of DNA from source cell. Organism DNA => pieces => put into vectors => plasmid or viruses => taken by host bacteria => Bacterial or bacteriophage clones contain all genes of that organisms.
Probe: To search genetic library for a certain gene. Single stranded => Nucleotide sequence => hybdrize with certain piece of DNA. Radioactive or flouroscent location possible.
After probe hybdrize into gene of interest => gene can be isolated.
PCR: 1983 => Kary B Mullis. Create million of copies of style gene or DNA => fastly intestube. Specific => million of total DNA => less man one part is tongetecl. Signal gene or DNA among as genes => can be copied using PCR. PCR name => from DNA polymerase => enzyme => DNA replication in cell. Chain reaction and million of copies of desired DNA.
Process: primers => sequence of 20 bases. Available must => complementary to bases on either side of target DNA. To start the replication process => DNA polymerase => extends process. DNA polymerase => Temperature insensitive or thermostable enzyme isolated => Termus aquatics in hot springs => Taq polymerase => DNA polymerase. Thermostable => sperate double stranded DNA => replication not interpret. Thermorecyclers => automatic PCR DNA fingerprinting.
RFLRs: Genome meclted with restriction enzyme => result in unique collection of different size fragment.
Gel Electrophoresis: Fragments separated according to length => number of bands DNA of single sperm => identify => rapist.
PCR amplification Uses: Diagone viral infections => hepatitis, genetic disorders, cancers. Identify criminals in forengic lab. evolutional history of human.
Gene Sequencing: Late 1970 => To determine Nucleotide sequence of DNA fragment at genes.
Principals: Generate different size DNA => start and ens point same. Separation of these pieces on a gasose Gel. Read sequence plan Gel. Two Methods: Sanger => dideoyribonucleoside triphosphate used to terminate DNA synthesis at different sites. Maxam Gilbert => DNA threads chemically cut in pieces of different size.
Genome sequence organisms.
Plant chloroplast or animal mitochondria. Bacterial, Dorsophilla.
Nematode.
Model plant Arabidopsis, human,mouse.
Human Genome project: By US government. Two goals • Genetic map of human genome.
• Contract base sequence map.
Genetic map of human genome => along lenght => each of chromosome type => human chromosomes 22 => smallest human chromosome. Human chromosomes => DNA sequence completed => 1999. Human chromosomes => see how genes are arranged along the vertebrate chromosome.
Entire human genome => 2002 publication.
Human genome => 25 times larger than any other sequence so fnal. RFLPs => sites to pinpoint disease causing gene bc² particular RFLP and defective gene => inhabited together.
E.g: Huntington disease.
Three billion base pairs of human genome. Instrumentation => J. Craig Ventor => company => Gelera => sequence entire genome.
Transgenic Organism:
Organism having foreign gene inserted into them. insulin, human growth hormone, haemophilia V111. Hepatitis B vaccine => produced by bacteria.
Bacteria => biofilters => prevent air borne chemical pollutants from being vented in air. Phenylalanine => organic chemical bto make aspartame dipeptide sweetener => Nutrasweet. Protoplast => Cellwall of plant removed. RuBP => capture corinne plants.
C⁴ cycle. Transgenic animal => insert in eggs of animals. Antithrombin 111 => prevents blood clot during surgery. Urine => preferable biotechnology product than milk Dolly Nancy => cloned => rollin Scotland => Cumulus cells => cling to egg after ovulation occurs.
Gene Therapy: Insertion of genetic material into human cell for treatment of disorder.
Two Methods: • In-vivo • Ex-vivo => SCID => Seven immune deficiency syndrome => lacks adenosine deaminase ADA thst matures B and T cells. Bone marrow stems tell preferred => divide and produce more cells with same gene.
Familial hyper Chloesterolemia => liver cells lacks receptor to remove chlestorol from blood. Cystic fibrosis => lack gene => thst codes transmembrane carrier of Cl ion. Infections of respiratory tract. Coronary artery angioplasty => ballons catheter => opens the closed artery => pi - galactosidase => enzyme => treat human lycosomal storage disease.
Tissue culture: Growth of tissue in artificial medium. Totipotent => 1902 => Gottleib => each cell had full gene potential => single gene => complete plant. Callux => Undifferentiated group if cells. Coconut milk => contains => cytokinin hormone. Mircopropagation => millions of identical seedlings in limited space. Meristem => fast growth => virus free. Anther culture => produce plants expressing recessive alleles. Enucleated egg =>egg that has its own nucleus removed.