Recombinant Technololgy Exploring Genes Recombinant Technololgy

Restriction Enzymes What are restriction enzymes and how are they used? enzymes that recognize specific base sequences in DNA and cleave the DNA at those sequences cuts DNA into specific fragments for analysis

Restriction Enzymes What characteristic do the cleavage sites of restriction enzymes share? palindromic

Restriction Enzymes How do we identify a particular restriction enzyme? based upon host organism from which enzyme was isolated first letter genus second two letters species

Restriction Enzymes What is a restriction digest and how is it analyzed? DNA cut by one or more restriction enzymes gel electrophoresis

Restriction Enzymes What is Southern blotting and how is it used? separating mixture of restriction fragments of DNA by electrophoresis and probing with labeled DNA

Restriction Enzymes What are Northern blots? What are Western blots? separation of RNA fragments and probing with labeled DNA What are Western blots? separation of proteins and probing with labeled antibody

DNA Sequencing How is controlled termination of DNA replication used to determine the sequence of a DNA molecule?

DNA Sequencing

DNA Sequencing How large are complete genomes? X174 virus – 5386 base pairs (bp) human mitocondrial DNA – 16, 569 bp Haemophilus influenzae – 1,830,137 bp C. elegans – 100,000,000 bp H. sapiens – 3,000,000,000 bp

DNA Synthesis How can DNA be synthesized in the lab? solid-phase synthesis by phosphite triester method

DNA Synthesis What does the activated monomer look like?

DNA Synthesis Why is the ability to synthesize DNA chains valuable? making radioactive or fluorescent probes helps to locate presence of genes synthetic probe can be used as primer can synthesize customized genes

Polymerase Chain Reaction What is PCR? technique used to make many copies of a specific DNA sequence Why is this a valuable tool? amplify a small amount of DNA What materials are needed? primers all dNTP’s heat-stable DNA polymerase

Polymerase Chain Reaction

Polymerase Chain Reaction What are some of the ways PCR has been used forensics paternity suits detection of HIV cancer detection detection of tuberculosis bacillus

Recombinant DNA What is recombinant DNA? novel DNA molecules What is needed to make recombinant DNA? vector plasmid virus

Recombinant DNA What else? restriction enzyme DNA ligase

Recombinant DNA

Recombinant DNA Any DNA molecule can be made to contain sticky or cohesive ends

Cloning Vectors Plasmids and bacteriophages serve as useful cloning vectors example – pBR322

Cloning Vectors Lambda phage can destroy host cell or become incorporated into host cell.

Cloning Vectors How are phages used as vectors? What advantages do these modified viruses have over plasmids?

Cloning Vectors What are some of the advantages of using M13 phage as a cloning vector? exists as a single-stranded molecule doesn’t kill bacterial host can grow large quantities of M13 can sequence foreign DNA cloned into M13 with dideoxy method

Cloning Vectors How is M13 used for cloning?

Recombinant DNA What is a genomic library and how is it prepared? collection of genomic DNA fragments inserted into a cloning vector

Recombinant DNA How does one screen a genomic library to find which phages contain a particular gene? grow phage on lawn of bacteria plaques form where phage infect cells prepare replica on nitrocellulose sheet treat with NaOH to lyse bacteria and denature DNA hybridize with 32P labeled probe audoradiography

Recombinant DNA What are cosmids? combinations of plasmids and lambda phages can hold 45 kb inserts What are bacterial artificial and yeast artificial chromosomes (BAC &YAC)? pieces of DNA containing centromere, autonomous replication sequence (ARS), and telomeres can hold 100-1000 kb inserts

Recombinant DNA What is chromosome walking and how is it used? technique of sub-cloning and re-screening used to analyze long stretches of DNA

Recombinant DNA What is c-DNA? DNA made from m-RNA How is it made?

Recombinant DNA How is c-DNA used? placed in vectors, inserted into bacteria, forms a c-DNA library How does a c-DNA library differ from a genomic DNA library? c-DNA contains no introns

Recombinant DNA How are c-DNA clones screened?

Recombinant DNA For what purpose are gene chips used? to measure the level of gene expression in eukaryotic cells via fluorescence

Recombinant DNA Why must many eukaryotic genes be expressed in eukaryotic cells? require posttranslational modification What is the most effective way of introducing eukaryotic DNA into host cells? retroviruses – Moloney murine leukemia virus Baculovirus – insect cells Vaccinia virus

Recombinant DNA What are transgenic mice? mice containing and expressing foreign DNA

Recombinant DNA What led to the formation of this oversized mouse?

Recombinant DNA What is homologous recombination and how has it been used to cause gene knockouts?

Recombinant DNA How can foreign genes be inserted into plant cells? Ti plasmids effective in dicots and a few monocots

Recombinant DNA How can foreign genes be inserted into plant cells? electroporation cereal monocots and dicots “gene guns” DNA coated onto tungsten pellets fired at cells

Recombinant DNA How can one construct a new gene by producing deletions? cut plasmid at two sites with restriction enzyme and ligate to form smaller plasmid cut plasmid at one site, use endonuclease to remove additional bases, and ligate

Recombinant DNA What is oligonucleotide-directed mutagenesis? used to replace code for particular amino acid prepare oligonucleotide primer containing a mutant code for amino acid to be changed separate plasmid strands, anneal primer, allow for replication some progeny will contain mutation

Recombinant DNA What is cassette mutagenesis? type of insertion

Recombinant DNA What are some of the benefits of this technology? understanding protein structure, mechanisms of catalysis, etc. produce large amounts of useful proteins insulin growth hormone drugs diagnostic reagents gene therapy agriculture