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
The Molecular Analysis of DNA, RNA, and
Protein
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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 (Gene
and plasmid).
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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
minichromosome.
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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 site-specific 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. (it is equal to its reverse complement)
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Structure of an EcoRI-DNA
Complex
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Many Restriction Endonucleases
Make Staggered (Sticky)Cuts
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When DNA is cleaved with a restriction
endonuclease that makes “sticky” 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)
“Vector" is an agent/system that can carry a DNA fragment
into a host cell.
If it is used for reproducing the DNA fragment, it is called a
"cloning vector".
If it is used for expressing certain gene in the DNA fragment, it
is called an "expression vector".
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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
(Phage lambda)
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 (lysogenic
pase-prophage-lysogen).
 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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Phagemid Vectors
Contain components from phage
chromosomes and plasmids (pBluescript II)
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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pBluescript II:
--hybrid (phage and plasmids)
--produce a vector that can grow
as
-----a plasmid
-----produce a single stranded
DNA in viral particles
--unique restriction sites
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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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Shuttle (Eukaryotic) 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 and it can propagate in two
different host species
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Importance/significance
Insulin
Antibodies
Growth Factors
Enzymes
The Polymerase Chain Reaction
(PCR)
 Synthetic nucleotides complementary to known flanking
sequences “fragment of nucleotides” are used to prime
enzymatic amplification of the sequence of interest “small
fragment of nucleotides.”
 Three repeated steps
– Denaturation of genomic DNA (92-95°C)
– Annealing of denatured DNA to oligonucleotide primers (50-60°C)
– Replication (Elongation) 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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nucleotides
1kb~1 min
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Synthesis of Double-Stranded cDNAs
from mRNA (also RT-PCR)
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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 ( e.g., insulin)
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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 (Cy5, Cy3) are used for
detection of DNA chains instead of
radioactive isotopes (32P ,35S).
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
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to a computer for analysis.
Automated Sanger DNA
Sequencing
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