Exploring Genes Recombinant Technololgy. Restriction Enzymes What are restriction enzymes and how...

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