Transcript Chapter 15 The Techniques of Molecular Genetics

Chapter 14
The Techniques of Molecular
Genetics
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Chapter Outline
Basic Techniques used to Identify, Amplify,
and Clone Genes
Construction and Screening of DNA Libraries
The Molecular Analysis of DNA, RNA, and
Protein
The Molecular Analysis of Genes and
Chromosomes
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Basic Techniques Used to
Identify, Amplify, and Clone
Genes
Recombinant DNA, gene cloning,
and DNA amplification techniques
allow scientists to isolate and
characterize essentially any DNA
sequence from any organism.
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Gene Cloning
Gene cloning is the isolation and
amplification of a given gene.
A recombinant DNA molecule is a
DNA molecule made by joining two or
more different DNA molecules.
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Amplification of a Gene In Vivo
A minichromosome carrying the gene of
interest is produced in the test tube.
The recombinant minichromosome is
introduced into a host cell (such as E.
coli), and the host cell replicates the
minochromosome.
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Amplification of a Gene In Vitro
Short DNA strands complementary to
DNA sequences on either side of the
gene of interest are synthesized.
These short DNA strands are used to
initiate the amplification of the gene by a
heat-stable DNA polymerase in the
polymerase chain reaction (PCR).
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Restriction Endonucleases
Restriction endonucleases make sitespecific cuts in DNA.
The nucleotide sequences are called
restriction sites.
Restriction endonucleases protect bacteria
from foreign DNA.
Bacteria protect endogenous restriction sites
by methylation.
Restriction enzymes commonly recognize
palindromic sequences.
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Structure of an EcoRI-DNA
Complex
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Many Restriction Endonucleases
Make Staggered Cuts
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When DNA is cleaved with a restriction
endonuclease that makes staggered cuts, all
of the resulting restriction fragments have
complementary single-stranded termini.
The complementary single-stranded termini
can hydrogen bond with each other and be
joined together by DNA ligase.
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Construction of Recombinant
DNA Molecules In Vitro
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Plasmid Vectors
Circular, double-stranded circular DNA
molecules present in bacteria.
Range from 1 kb to over 200 kb.
Replicate autonomously.
Many carry antibiotic-resistance genes, which
can be used as selectable markers.
Many useful cloning vectors were derived
from plasmid pBR322.
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Bacteriophage Vectors
Most bacteriophage cloning vectors have
been constructed from the phage 
chromosome.
The central one-third (about 15 kb) of the 
chromosome contains genes required for
lysogeny but not for lytic growth.
This portion of the chromosome can be
excised and replaced with foreign DNA.
The foreign DNA inserted must be 10-15 kb.
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Cosmid Vectors
 Hybrids between plasmids and the phage 
chromosome.
 Replicate autonomously in E. coli.
 Can be packaged in vitro into phage  heads.
 Accept inserts of 35-45 kb.
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Phagemid Vectors
Contain components from phage
chromosomes and plasmids.
Replicate in E. coli as double-stranded
plasmids.
Addition of a helper phage causes the
phagemid to switch to the phage mode of
replication, resulting in the packaging of
single-stranded DNA into phage heads.
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The Blue-White Color Test
 The E. coli lacZ gene
encodes -galactosidase.
 -galactosidase converts
the colorless substrate
Xgal into a blue product.
 Cells with -galactosidase
activity produce blue
colonies when grown on
Xgal; cells lacking galactosidase activity
produce white colonies.
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Eukaryotic and Shuttle Vectors
Because different organisms use different
origins of replication and regulatory signals,
different cloning vectors must be used in
different species.
Special cloning vectors can replicate in other
prokaryotes and in eukaryotes.
Shuttle vectors can replicate in E. coli and in
another species.
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Yeast Artificial Chromosomes
(YACs)
Genetically engineered yeast
minichromosomes.
Accept foreign DNA inserts of 200-500
kb.
Contain a yeast origin of replication,
yeast centromere, two yeast telomeres,
a selectable marker, and a polycloning
site.
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BACs and PACs
Bacterial artificial chromosomes (BACs) have
been constructed from bacterial fertility (F)
factors.
Bacteriophage P1 artificial chromosomes
(PACs) have been constructed from
bacteriophage P1 chromosomes.
BACs and PACs accept 150-300 kb inserts
and are less complex than YACs.
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The Polymerase Chain
Reaction (PCR)
 Synthetic nucleotides complementary to known
flanking sequences are used to prime enzymatic
amplification of the sequence of interest.
 Three repeated steps
– Denaturation of genomic DNA (92-95°C)
– Annealing of denatured DNA to oligonucleotide primers (5060°C)
– Replication of the DNA segment between the sites
complementary to the primers (70-72°C)
 Amplification occurs exponentially; each cycle
doubles the number of molecules of the sequence of
interest.
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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.
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Applications of PCR
Diagnosis of inherited human diseases
(e.g., prenatal diagnosis).
Identification of individuals in forensic
cases from small DNA samples.
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Key Points
• The discovery of restriction endonucleases—
enzymes that recognize and cleave DNA in a
sequence-specific manner—allowed
scientists to produce recombinant DNA
molecules in vitro.
• DNA sequences can be inserted into small,
self-replicating DNA molecules called cloning
vectors and amplified by replication in vivo
after being introduced into living cells by
transformation.
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Key Points
• The polymerase chain reaction (PCR)
can be used to amplify specific DNA
sequences in vitro.
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Construction and Screening of
DNA Libraries
DNA libraries can be constructed
and screened for genes and other
sequences of interest.
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DNA Libraries
A genomic DNA library is a set of DNA
clones collectively containing the entire
genome of an organism.
A cDNA library contains the coding regions
of the expressed genes of an organism. It is
made of complementary DNA (cDNA)
synthesized from RNA by reverse
transcriptase.
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Cloning Restriction Fragments with
Complementary Single-Stranded Ends
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Amplification of Recombinant DNA
Antibiotic-sensitive recipient cells are
transformed with the recombinant DNA
molecule.
Transformed cells are selected by growth
under conditions requiring the presence of a
selectable marker present on the
recombinant DNA molecule (usually an
antibiotic).
The recombinant DNA molecule is amplified
by the host cell.
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Synthesis of Double-Stranded
cDNAs from mRNA
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Screening DNA Libraries for
Genes of Interest
Genetic Selection—searching for a
DNA sequence that restores the wildtype phenotype to a mutant organism.
Molecular hybridization is based on
the hybridization of similar DNA
sequences.
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Colony
Hybridization
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Key Points
• DNA libraries can be constructed that contain
complete sets of genomic DNA sequences or
DNA copies (cDNAs) of mRNAs in an
organism.
• Specific genes or other DNA sequences can
be isolated from DNA libraries by genetic
complementation or by hybridization to labeled
nucleic acid probes containing sequences of
known function.
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The Molecular Analysis of
DNA, RNA, and Protein
DNA, RNA, or protein molecules
can be separated by gel
electrophoresis, transferred to
membranes, and analyzed by
various procedures.
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Analysis of DNAs by Southern
Blot Hybridization
DNA molecules can be separated by size by
gel electrophoresis using agarose or
acrylamide gels for larger and small DNA
molecules, respectively.
DNA molecules can then be transferred from
the gel onto a nitrocellulose or nylon
membrane using a technique called a
Southern blot.
DNA on the membrane can be hybridized
with DNA probes.
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Agarose Gel Electrophoresis
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Southern Blot:
Transferring DNA from the Gel to a
Membrane
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Identification of a Specific Fragment
by Southern Blot Hybridization
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Detection of Wild-Type and Mutant
Alleles of the Cystic Fibrosis Gene
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Analysis of RNAs by Northern
Blot Hybridizations
The Northern Blot procedure is nearly
identical to Southern blotting, except
– RNA is sensitive to degradation by RNases;
contamination with these enzymes must be
prevented.
– RNA molecules contain extensive secondary
structure and must be kept denatured during
electrophoresis.
Northern blots are useful in studies of gene
expression.
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Northern Blot Hybridization Data
(RNA from roots, leaves, and flowers of A. thaliana)
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Analysis of RNAs by Reverse
Transcriptase-PCR (RT-PCR)
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Analysis of Proteins by
Western Blot Techniques
Polypeptides are separated by
polyacrylamide gel electrophoresis in
the presence of a detergent that
denatures the proteins.
Proteins are transferred from the gel to
a nitrocellulose membrane.
Individual proteins are detected with
antibodies.
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Key Points
• DNA restriction fragments and other small
DNA molecules can be separated by agarose
or acrylamide gel electrophoresis and
transferred to nylon membranes to produce
DNA gel blots called Southern blots.
• The DNAs on Southern blots can be
hybridized to labeled DNA probes to detect
sequences of interest by autoradiography.
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Key Points
• When RNA molecules are separated by gel
electrophoresis and transferred to
membranes for analysis, the resulting RNA
gel blots are called northern blots.
• RNA molecules can be detected and
analyzed by reverse transcriptase-PCR (RTPCR).
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Key Points
• When proteins are transferred from gels to
membranes and detected with antibodies, the
products are called western blots.
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The Molecular Analysis of
Genes and Chromosomes
The sites at which restriction enzymes
cleave DNA molecules can be used to
construct physical maps of the molecules;
however, nucleotide sequences provide
the ultimate physical maps of DNA
molecules.
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Mapping Restriction Enzyme
Cleavage Sites
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Restriction maps reflect true physical
distances (unlike genetic maps).
Restriction maps can be combined with
other molecular techniques to construct
physical maps of entire genomes.
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Techniques Necessary for
Sequencing DNA
 Restriction enzymes to prepare homogenous
samples of specific segments of chromosomes.
 Gel electrophoresis procedures able to resolve DNA
fragments differing in length by a single nucleotide.
 Gene-cloning techniques allowing preparation of
large quantities of a DNA molecule.
 Sanger sequencing Technique is used to determine
nucleotide sequences.
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DNA Sequencing
A population of DNA fragments is
generated.
– One end is common to all fragments (the 5’
end of the sequencing primer).
– The other end terminates at all possible
positions (the 3- terminus).
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2',3'-Dideoxyribonucleoside
Triphosphates
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Automated DNA Sequencing
Fluorescent dyes are used for detection of
DNA chains instead of radioactive isotopes.
Products of all four chain terminator reactions
are separated through a single gel or capillary
tube.
Photocells detect fluorescence of the dyes as
they pass through the gel or capillary tube.
Output of the photocell is directly transferred
to a computer for analysis.
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Automated Sanger DNA
Sequencing
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Key Points
• Detailed physical maps of DNA
molecules can be prepared by
identifying the sites that are cleaved by
various restriction endonucleases.
• The nucleotide sequences of DNA
molecules provide the ultimate physical
maps of genes and chromosomes.
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