exploring genes recombinant technololgy. restriction enzymes what are restriction enzymes and how...
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Exploring Genes
Recombinant Technololgy
Restriction EnzymesWhat 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
What are Northern blots? separation of RNA fragments and probing
with labeled DNA
What are Western blots? separation of proteins and probing with
labeled antibody
Restriction Enzymes
DNA Sequencing How is controlled termination of DNA
replication used to determine the sequence of a DNA molecule?
DNA Sequencing
DNA SequencingHow 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 SynthesisWhy 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 ReactionWhat are some of the ways PCR has
been used forensics paternity suits detection of HIV cancer detection detection of tuberculosis bacillus
Recombinant DNAWhat is recombinant DNA?
novel DNA moleculesWhat is needed to make recombinant
DNA? vector
plasmid virus
Recombinant DNAWhat 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 VectorsWhat 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 DNAHow 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 DNAHow 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 DNAWhat 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