Unit II Lecture 3 B. Tech. (Biotechnology) III Year V th Semester EBT-501, Genetic Engineering

EBT 501, Genetic Engineering Unit I Gene cloning -concept and basic steps; application of bacteria and viruses in genetic engineering; Molecular biology of E. coli and bacteriophages in the context of their use in genetic engineering, Cloning vectors: Plasmid cloning vector PBR322, Vectors for cloning large piece of DNA; –Bacteriophage-l and other phage vectors; Cosmids, Phagemids; YAC and BAC vectors, Model vectors for eukaryotes – Viruses, Unit II Restriction modification, enzymes used in recombinant DNA technology endonucleases, ligases and other enzymes useful in gene cloning, PCR technology for gene/DNA detection, cDNA, Use of Agrobacterium for genetic engineering in plants; Gene libraries; Use of marker genes. Cloning of foreign genes: DNA delivery methods -physical methods and biological methods, Genetic transformation of prokaryotes: Transferring DNA into E. coli –Chemical induction and Electroporation, Unit III Gene library: Construction cDNA library and genomic library, Screening of gene libraries – screening by DNA hybridization, immunological assay and protein activity, Marker genes: Selectable markers and Screenable markers, nonantibiotic markers, Gene expression in prokaryotes: Tissue specific promoter, wound inducible promoters, Strong and regulatable promoters; increasing protein production; Fusion proteins; Translation expression vectors; DNA integration into bacterial genome; Increasing secretions; Metabolic load, Recombinant protein production in yeast: Saccharomyces cerevisiae expression systems; Mammalian cell expression vectors: Selectable markers; Unit IV Origins of organismal cloning in developmental biology research on frogs; nuclear transfer procedures and the cloning of sheep (Dolly) & other mammals; applications in conservation; therapeutic vs. reproductive cloning; ethical issues and the prospects for human cloning; Two-vector expression system; two-gene expression vector, Directed mutagenesis; transposon mutagenesis, Gene targeting, Site specific recombination Unit V General principles of cell signaling, Extracellular signal molecule and their receptors, Operation of signaling molecules over various distances, Sharing of signal information, Cellular response to specific combinations of extracellular signal molecules; Different response by different cells to same extracellular signal molecule, NO signaling by binding to an enzyme inside target cell, Nuclear receptor; Ion channel linked, G-protein- linked and enzyme-linked receptors, Relay of signal by activated cell surface receptors via intracellular signaling proteins, Intracellular signaling proteins as molecular switches, Interaction between modular binding domain and signaling proteins, Remembering the effect of some signal by cells.

PCR PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. It is called “chain” because the products of the first reaction become substrates of the following one, and so on. From a single copy of DNA, the PCR can generate billions of copies of specific DNA sequences by the simultaneous primer extension of complementary DNA strands. The reaction is a cyclic process, which is repeated times. One cycle consists of three basic steps.

PCR Steps Synthetic nucleotides complementary to known flanking sequences are used to prime enzymatic amplification of the sequence of interest. Denaturation of the double stranded DNA to make the template available for the primers and DNA polymerase. It is done at temperature 94 o C for 1minute. Annealing of primers to complementary sequence on template. It depend upon the type of primers used. It varies considerably from as low as 37 o C for RAPD primers to as high as 60 o C for some GC rich primers. Extension of primers by DNA polymerase (72 o C is the optimum temperature of Taq DNA Polymerase. Amplification occurs exponentially; each cycle doubles the number of molecules of the sequence of interest.

Purpose of PCR Amplify large quantities of DNA (μg quantities) from small quantities (fg quantities) [billion fold amplification] Analyze single DNA fragments out of large complex mixture. Alter DNA sequence – directed mutagenesis. PCR is commonly used to… Identify species Identify alleles/genotypes to assess variability in a population Create sequences for phylogenies to determine taxonomic relationships. Conduct forensic investigations PCR is NOT used to: Amplify RNA or proteins Construct genomic or cDNA libraries Make monoclonal antibodies Conduct stem cell research

ComponentQuantityFinal Concentration Sterile Water12.8 µl 10 x PCR Buffer5 µl1 x 15 mM MgCl 2 5 µl1.5 mM 1 mM dNTP mix10 µl0.2 mM 5 µM forward primer2.5 µl0.25 µM 5 µM reverse primer2.5 µl0.25 µM 5 U/µl Taq polymerase0.2 µl1 U/Reaction 25 ng/µl template DNA 2 µl50-100ng Total50.0 µl Laboratory PCR Steps Create Master Mix (Table 1) of reagents and aliquot into tubesAdd DNA template(s) Program thermal cycler (Table 2), load with tubes and start Remove tubes and analyze results Table 1. Master Mix and Addition of template DNA Laboratory PCR Steps Create Master Mix of reagents and aliquot into tubes Add DNA template(s) Program thermal cycler, load with tubes and start Remove tubes and analyze results

Taq Polymerase DNA polymerase from Thermus aquaticus is used for PCR because it is heat-stable. Taq polymerase lacks proofreading activity, so errors are introduced into the amplified DNA at low but significant frequencies. –When high fidelity is required, heat-stable polymerases with proofreading activity are used (Pfu or Tli). Taq is amplifies fragments of DNA larger than a few thousand base pairs inefficiently. –For amplification of long segments of DNA (up to 35 kb), Tfl polymerase is used.

Applications of PCR Molecular Identification Molecular Archaeology Bioinformatics Site directed mutagenesis Molecular Epidemiology Genomic cloning Gene expression studies Molecular Ecology Human Genome Project

Applications of PCR Sequencing Bioinformatics Genomic cloning Human Genome Project Genetic Engineering Site directed mutagenesis Gene expression studies

Other Applications of PCR Detection of chromosomal translocations –Amplification across a translocation sequence Chromosome painting Detection of residual disease Infectious disease Forensics HLA typing Detection of Loss of Suppressor Genes –Loss of Heterozygosity (LOH)