Download Pierce5e_ch19_lecturePPT

Benjamin A. Pierce
GENETICS
A Conceptual Approach
FIFTH EDITION
CHAPTER 19
Molecular Genetic Analysis and
Biotechnology
© 2014 W. H. Freeman and Company
Techniques of Molecular Genetics Have
Revolutionized Biology
• Recombinant DNA Technology (Genetic
Engineering)
– Techniques for locating, isolating, altering, and
studying DNA segments
• The Molecular Genetics Revolution
– Biotechnology: the use of these techniques to
develop new products
• Working at the Molecular Level
Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• First step: isolate DNA segment or gene from
remaining DNA
• Cutting and joining DNA fragments—restriction
enzymes
• Viewing DNA fragments
• Locating DNA fragments with southern blotting
and probes
Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• First step: isolate DNA segment or gene from
remaining DNA
• Cutting and joining DNA fragments—restriction
enzymes
• Viewing DNA fragments
• Locating DNA fragments with southern blotting
and probes
Cutting and Joining DNA Fragments
• Restriction enzymes: recognizing and cutting
DNA at specific nucleotide sequences
• Type II restriction enzyme: most useful enzyme
• By adding methyl groups to the recognition
sequence to protect itself from being digested by
its own enzyme in bacteria
Cutting and Joining DNA Fragments
• Cohesive ends: fragments with short, singlestranded overhanging ends
• Blunt ends: even-length ends from both single
strands
Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• First step: isolate DNA segment or gene from
remaining DNA
• Cutting and joining DNA fragments—restriction
enzymes
• Viewing DNA Fragments
• Locating DNA fragments with southern blotting
and probes
Viewing DNA Fragments
• Gel electrophoresis
• Autoradiography
Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• First step: isolate DNA segment or gene from
remaining DNA
• Cutting and joining DNA fragments—restriction
enzymes
• Viewing DNA fragments
• Locating DNA fragments with southern blotting
and probes
Locating DNA Fragments with
Southern Blotting and Probes
• Probe: DNA or RNA with a base sequence
complementary to a sequence in the gene of
interest
Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• Cloning genes
• Application: the genetic engineering of plants
with pesticides
• Amplifying DNA fragments with the polymerase
chain reaction
Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• Cloning genes
• Application: the genetic engineering of plants
with pesticides
• Amplifying DNA fragments with the polymerase
chain reaction
Cloning Genes
• Gene cloning: amplifying a specific piece of
DNA via a bacteria cell
• Cloning vector: a replicating DNA molecule
attached with a foreign DNA fragment to be
introduced into a cell
Cloning Genes
• Plasmid vectors
– Plasmids: circular DNA molecules from bacteria
– Insert foreign DNA into plasmid using restriction
enzymes
– Linkers: synthetic DNA fragments containing
restriction sites
• Transformation of host cells with plasmids
• Screening cells for recombinant plasmids
– Selectable markers are used to confirm whether the
cells have been transformed or not
Cloning Genes
• Other gene vectors:
–
–
–
–
Cosmids
Bacterial Artificial Chromosomes (BACs)
Yeast Artificial Chromosome
Ti plasmid
Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• Cloning genes
• Application: the genetic engineering of plants
with pesticides
• Amplifying DNA fragments with the polymerase
chain reaction
Application: The Genetic Engineering of
Plants with Pesticide
• Bacillus thuringienis
• Bt gene
• Agrobacterium tumefaciens
Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• Cloning genes
• Application: the genetic engineering of plants
with pesticides
• Amplifying DNA fragments with the polymerase
chain reaction
Amplifying DNA Fragments with the
Polymerase Chain Reaction (PCR)
• The PCR reaction
– Taq polymerase: stable DNA polymerase at high
temperature
– Reverse-transcription PCR
Amplifying DNA Fragments with the
Polymerase Chain Reaction (PCR)
• Limitations of PCR
– Prior knowledge of target DNA
– Contamination
– Accuracy
– Amplified fragments are less than 2 kb
Amplifying DNA fragments with the
polymerase chain reaction (PCR)
• Applications of PCR
– Real-time PCR: quantitatively determining the
amount of DNA amplified as the reaction proceeds
Molecular Techniques Can Be Used to Find
Genes of Interest
• Gene libraries
• In situ hybridization
• Positional cloning
• Application: isolating the gene for cystic fibrosis
Molecular Techniques Can Be Used to Find
Genes of Interest
• Gene libraries
• In situ hybridization
• Positional cloning
• Application: isolating the gene for cystic fibrosis
Gene Libraries
• DNA library: a collection of clones containing all
the DNA fragments from one source
– Creating a genomic DNA library
– cDNA libraries: consisting only of those DNA
sequences that are transcribed into mRNA
Gene Libraries
• Screening DNA libraries
– Plating clones of the library
– Probing plated colonies or plaques
Molecular Techniques Can Be Used to Find
Genes of Interest
• Gene libraries
• In situ hybridization
• Positional cloning
• Application: isolating the gene for cystic fibrosis
In Situ Hybridization
• DNA probes used to determine the
chromosomal location and to visualize a gene
while it is in a cell
• FISH
Molecular Techniques Can Be Used to Find
Genes of Interest
• Gene libraries
• In situ hybridization
• Positional cloning
• Application: isolating the gene for cystic fibrosis
Positional Cloning
• Isolating genes on the basis of their position on
a genetic map
• Chromosome walking
• Chromosome jumping
Molecular Techniques Can Be Used to Find
Genes of Interest
• Gene libraries
• In situ hybridization
• Positional cloning
• Application: isolating the gene for cystic fibrosis
Application: Isolating the Gene for Cystic
Fibrosis
• Autosomal recessive disorder
• Characterized by chronic lung infections,
insufficient pancreatic enzyme production, and
increased salt concentration in sweat
DNA Sequences Can Be Determined and
Analyzed
• Restriction Fragment Length Polymorphisms
(RFLPs)
• DNA sequencing
• Next-generation sequencing technologies
• DNA fingerprinting
• Application: identifying people who died in the
collapse of the World Trade Center
DNA Sequences Can Be Determined and
Analyzed
• Restriction Fragment Length Polymorphisms
(RFLPs)
• DNA sequencing
• Next-generation sequencing technologies
• DNA fingerprinting
• Application: identifying people who died in the
collapse of the World Trade Center
Restriction Fragment Length Polymorphisms
• Some DNA fragments have different restriction
sites due to mutation for the same restriction
enzyme
• Causes polymorphisms within a population
DNA Sequences Can Be Determined and
Analyzed
• Restriction Fragment Length Polymorphisms
(RFLPs)
• DNA sequencing
• Next-generation sequencing technologies
• DNA fingerprinting
• Application: identifying people who died in the
collapse of the World Trade Center
DNA Sequencing
• Sanger’s dideoxy-sequencing method
– Dideoxyribonucleoside triphosphate (ddNTP)
lacks a 3′-oh group, which terminates DNA
synthesis
DNA Sequences Can Be Determined and
Analyzed
• Restriction Fragment Length Polymorphisms
(RFLPs)
• DNA sequencing
• Next-generation sequencing technologies
• DNA fingerprinting
• Application: identifying people who died in the
collapse of the World Trade Center
Next-Generation Sequencing Technologies
• Pyrosequencing
• Illumina sequencing
• Third-generation sequencing
DNA Sequences Can Be Determined and
Analyzed
• Restriction Fragment Length Polymorphisms
(RFLPs)
• DNA sequencing
• Next-generation sequencing technologies
• DNA fingerprinting
• Application: identifying people who died in the
collapse of the World Trade Center
DNA Fingerprinting (DNA Profiling)
• Microsatellites: (short tandem repeats, STRs)
variable number of copies of repeat sequences
possessed by many organisms
• Detected by PCR
• Fragments represented as peaks on a graph
– Homozygotes: single tall peak
– Heterozygotes: two shorter peaks
DNA Sequences Can Be Determined and
Analyzed
• Restriction Fragment Length Polymorphisms
(RFLPs)
• DNA sequencing
• Next-generation sequencing technologies
• DNA fingerprinting
• Application: identifying people who died in the
collapse of the World Trade Center
Application: Identifying People Who Died in
the Collapse of the World Trade Center
• Usual means of victim
ID were of little use
with WTC remains
• Used DNA
fingerprinting
• Also carried out on
mitochondrial DNA
• INSERT FIG 19.31
Molecular Techniques Are Increasingly Used
to Analyze Gene Function
•
•
•
•
•
•
•
Forward and reverse genetics
Creating random mutations
Site-directed mutagenesis
Transgenic animals
Knockout mice
Silencing genes with RNAi
Application: Using RNAi for the treatment of
human disease
Forward and Reverse Genetics
• Forward genetics: Begins with a phenotype to
a gene that encodes the phenotype
• Reverse genetics: Begins with a gene of
unknown function, first inducing mutations
and then checking the effect of the mutation
on the phenotype
Creating Random Mutations
• A means to increase the number of mutants in
an experimental population
• Use mutagenic agents: radiation, chemical
mutagens, transposable elements
Site-Directed Mutagenesis
• Site-directed mutagenesis
• Oligonucleotide-directed mutagenesis
Transgenic Animals
• An organism permanently altered by the addition
of a DNA sequence to its genome
• Transgene
Knockout Mice
• A normal gene of the mouse has been fully
disabled
• Knock-in mice: a mouse carries an inserted
DNA sequence at specific locations
Silencing Genes with RNAi
• siRNAs
• Process called RNA interference (RNAi)
Application: Using RNAi for the Treatment
of Human Disease
• High cholesterol
• RNAi could be used to reduce the levels of
ApoB and blood cholesterol in nonhuman
primates
Biotechnology Harnesses the Power
of Molecular Genetics
• Pharmaceutical products
• Specialized bacteria
• Agriculture products
• Genetic testing
• Gene therapy
Concept Check 1
A geneticist is interested in the immune function
of mice and induces random mutations in a
number of genes in mice and then determines
which of the resulting mutant mice have
impaired immune function. This is an example of
________.
a.
b.
c.
d.
forward genetics
reverse genetics
both forward and reverse genetics
neither forward nor reverse genetics
Concept Check 1
A geneticist is interested in the immune function
of mice and induces random mutations in a
number of genes in mice and then determines
which of the resulting mutant mice have
impaired immune function. This is an example of
________.
a.
b.
c.
d.
forward genetics
reverse genetics
both forward and reverse genetics
neither forward nor reverse genetics