Download Section 20.1

Chapter 20 Lecture
Concepts of Genetics
Tenth Edition
Recombinant DNA
Technology
Chapter Contents
20.1
20.2
20.3
20.4
20.5
Recombinant DNA Technology Began with Two Key Tools:
Restriction Enzymes and DNA Cloning Vectors
DNA Libraries Are Collections of Cloned Sequences
The Polymerase Chain Reaction Is a Powerful Technique for
Copying DNA
Molecular Techniques for Analyzing DNA
DNA Sequencing Is the Ultimate Way to Characterize DNA
Structure at the Molecular Level
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20.1 Recombinant DNA Technology
Began with Two Key Tools: Restriction
Enzymes and DNA Cloning Vectors
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Section 20.1
• Recombinant DNA refers to the joining of DNA
molecules, usually from different biological
sources, that are not found together in nature
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Section 20.1
• The basic procedure for producing recombinant
DNA involves
– generating specific DNA fragments using restriction
enzymes
– joining these fragments with a vector
– transferring the recombinant DNA molecule to a host
cell to produce many copies that can be recovered
from the host cell
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Section 20.1
• The recovered copies of a recombinant DNA molecule
are referred to as clones and can be used to study the
structure and orientation of the DNA
• Recombinant DNA technology is used to isolate,
replicate, and analyze genes
• A restriction enzyme binds to DNA at a specific
recognition sequence (restriction site) and cleaves the
DNA to produce restriction fragments
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Section 20.1
• Most recognition sequences exhibit a form of symmetry
described as a palindrome, and restriction enzymes cut
the DNA in a characteristic cleavage pattern
– Mostly four to six nucleotides long
– Some contain eight or more nucleotides
• DNA ligase joins restriction fragments covalently to
produce intact DNA molecules
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Section 20.1
• Vectors are carrier DNA molecules that can replicate
cloned DNA fragments in a host cell
• Vectors must be able to replicate independently and
should have several restriction enzyme sites to allow
insertion of a DNA fragment
• Vectors should carry a selectable gene marker to
distinguish host cells that have taken them up from those
that have not
• A plasmid is an extrachromosomal double-stranded
DNA molecule that replicates independently from the
chromosomes within bacterial cells
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Section 20.1
• Plasmids used for DNA cloning usually have been
engineered to contain
– a number of convenient restriction sites
– a marker gene to select for its presence in the host cell
• Plasmids are introduced by the process of transformation
• Transformation is achieved through
– using calcium ions and brief heat shock to pulse DNA into cells
– Electroporation, which uses a brief but high-intensity pulse of
electricity to move DNA into bacterial cells
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Section 20.1
• Both the plasmid DNA and DNA to be cloned are cut
with the same restriction enzyme
• DNA restriction fragments from the DNA to be cloned are
added to the linearized vector in the presence of DNA
ligase
• A recombinant DNA is produced, which is then
introduced into bacterial host cells by transformation
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Section 20.1
• Recombinant DNA can be readily identified by using
selectable marker genes
• Genes that provide resistance to antibiotics such as
ampicillin and genes such as the lacZ genes are very
effective selectable markers
• Blue-white selection is used to identify cells containing
recombinant and nonrecombinant DNA
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Section 20.1
• Phage vectors were among the earliest vectors used in
addition to plasmids
• The central third of lambda (λ) phage vectors can be
replaced with foreign DNA without affecting the ability to
infect cells and replicate
• Lambda vectors can carry up to 45 kb of cloned DNA
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Section 20.1
• Bacterial artificial chromosomes (BACs) and yeast
artificial chromosomes (YACs) are two other examples
of vectors that can be used to clone large fragments of
DNA
• BACs are generally very large but low copy number (one
to two copies/bacterial cell) plasmids
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Section 20.1
• Expression vectors are engineered to express a gene of
interest to produce large quantities of the encoded protein
in a host cell
• Expression vectors are available for both prokaryotic and
eukaryotic host cells
• Plant and animal cells can serve as hosts for recombinant
DNA, in addition to bacteria and yeast
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Section 20.1
• Rhizobium radiobacter (formerly Agrobacterium
tumefaciens) can be used to transform plant cells with TDNA containing foreign DNA
• Rhizobium contains the Ti plasmid (tumor inducing) with
genes that induce tumors
• Tumor-inducing genes can be removed from the vector
so the recombinant vector does not produce tumors
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Section 20.1
• The vector when mixed with plant cells enters inside the
cell, and the foreign DNA gets inserted into the plant
genome
• The plant cells can be grown in tissue culture and
eventually regenerate a mature plant carrying a foreign
gene
• These cells are then grown by tissue culture to give a
mature plant carrying a foreign gene
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Section 20.1
• While E. coli is used as a prokaryotic host cell when
working with plasmids, yeast is widely used as a host for
DNA cloning and expression of eukaryotic genes
because
–
–
–
–
–
it can be grown easily and manipulated
its genetics have been studied intensively
its entire genome has been sequenced
it can posttranslationally modify eukaryotic proteins
it is considered to be safe
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Section 20.1
• A variety of different human cell types can be grown in
culture and used to express genes and proteins
• These lines can be subjected to various approaches for
gene or protein functional analysis, including drug testing
for effectiveness at blocking or influencing a particular
recombinant protein being expressed, especially if the
cell lines are of a human disease condition such as
cancer
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20.2 DNA Libraries Are Collections of
Cloned Sequences
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Section 20.2
• DNA libraries represent a collection of cloned DNA samples derived
from a single source that could be a particular tissue type, cell type,
or single individual
• A genomic library contains at least one copy of all the sequences
in the genome of interest
• Genomic libraries are constructed by cutting genomic DNA with a
restriction enzyme and ligating the fragments into vectors, which are
chosen depending on the size of the genome
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Section 20.2
• YACs were commonly used to accommodate large sizes of DNA
necessary to span the 3 billion bp of DNA in the human genome
• Whole-genome shotgun cloning approaches and new sequencing
methodologies (next-generation sequencing) are replacing
traditional genomic libraries
• All DNA fragments in a genomic sample are sequenced without the
need to insert DNA fragments into vectors and cloning them in host
cells
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Section 20.2
• Complementary DNA (cDNA) libraries contains
complementary DNA copies made from the mRNAs
present in a cell population and represents the genes that
are transcriptionally active at the time the cells were
collected for mRNA isolation
• A cDNA library is prepared by
– isolating mRNA from cells
– synthesizing the complementary DNA using reverse transcriptase
– cloning the cDNA molecules into a vector
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Section 20.2
• Reverse transcriptase PCR (RT-PCR) can be used to
generate cDNA from mRNA by
– first making a single-stranded cDNA copy of the mRNAs using
reverse transcriptase
– then using PCR to copy the single-stranded DNA into doublestranded DNA
• The cDNA libraries provide an instant catalog of all the
genes active in a cell at a specific time and have been
very valuable tools for scientists isolating and studying
genes in a particular tissue
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Section 20.2
• Library screening is used to sort through a library and
isolate specific genes of interest
• Probes are used to screen a library to recover clones of
a specific gene. A probe is any DNA or RNA sequence
that is complementary to the target gene of sequence to
be identified
• To screen a plasmid library, clones from the library are
grown on agar plates to produce colonies. The colonies
are screened by transferring bacterial colonies from the
plate to a filter and hybridizing the filter with a nucleic
acid probe to the DNA sequence of interest
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Section 20.2
• The colony corresponding to the one the probe identified
on the filter is identified and recovered
• A phage library is screened by plaque hybridization
• Libraries enable scientists to clone DNA and then
identify individual genes in the library
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20.3 The Polymerase Chain Reaction Is a
Powerful Technique for Copying DNA
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Section 20.3
• The polymerase chain reaction (PCR) copies a
specific DNA sequence through in vitro reactions that
can amplify target DNA sequences present in very small
quantities
• PCR is a rapid method of DNA cloning that eliminates
the need to use host cells for cloning
• The double-stranded DNA to be cloned is put in a tube
with DNA polymerase, Mg2+, and the four dNTPs
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Section 20.3
• PCR requires two oligonucleotide primers (short,
single-stranded sequences), one complementary to the
5' end of one strand of the target DNA and another
complementary to the 3' end of the other strand
• The primers anneal to denatured DNA, and the
complementary strands are synthesized by a heat-stable
DNA polymerase
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Section 20.3
• The three steps of PCR—denaturation, primer annealing,
and extension—are repeated over and over using a
thermocycler to amplify the DNA exponentially
• The DNA strand is doubled in each cycle, and the new
strands along with the old strand serve as templates in
the next cycle
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Section 20.3
• A limitation of PCR is that some information about the
nucleotide sequence of the target DNA must be known in
order to synthesize the primer
• Minor contamination from other sources can cause
problems
• PCR cannot amplify long segments of DNA
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Section 20.3
• PCR is a very useful tool since it allows the screening of
mutations involved in in genetic disorders
• The location and nature of a mutation can be determined
quickly
• Allele-specific probes for genetic testing can be synthesized;
PCR is important for diagnosing genetic disorders
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Section 20.3
• PCR techniques are particularly advantageous when
studying samples from single cells, fossils, or a crime
scene, where a single hair or even a saliva-moistened
postage stamp in the source of DNA
• PCR has been used to enforce the worldwide ban on the
sale of certain whale products and determination of
pedigree background of purebred dogs
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Section 20.3
• Reverse transcription PCR (RT-PCR) is used to study
gene expression by studying mRNA production by cells
or tissues
• Quantitative real-time PCR (qPCR) or real-time PCR
allows researchers to quantify amplification reactions as
they occur in ‘real time’
– The procedure uses an SYBR green dye and TaqMan probes,
which contain two dyes
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20.4 Molecular Techniques for
Analyzing DNA
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Section 20.4
• A restriction map establishes the number and order of
restriction sites and the distance between restriction
sites on a cloned DNA segment
• It provides information about the length of the cloned
insert and the location of restriction sites within the clone
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Section 20.4
• Restriction maps were created by cutting DNA with
different restriction enzymes and separating the DNA
fragments by gel electrophoresis, which separates
fragments by size
• The smallest fragments move farthest in the gel
• These fragments can be visualized when stained with
ethidium bromide and illuminated by UV light
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Section 20.4
• A Southern blot is used to identify which clones in a
library contain a given DNA sequence and to
characterize the size of the fragments from restriction
digest
• Southern blots can also be used to
– determine whether a clone contains all or part of a gene
– ascertain the size and sequence organization of a gene or DNA
sequence of interest
• Southern blot has two components: separation of DNA
fragments by gel electrophoresis and hybridization by
using labeled probes
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Section 20.4
• Northern blot analysis is used to determine whether a
gene is actively being expressed in a given cell or tissue
– Used to study patterns of gene expression in embryonic tissues,
cancer, and genetic disorders
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Section 20.4
• Fluorescent in situ hybridization (FISH) involves
hybridizing a probe directly to a chromosome or RNA
without blotting
• FISH can be carried out with isolated chromosomes on a
slide or in situ in tissue sections or entire organisms
• FISH is especially helpful when embryos are used for
various studies in developmental genetics
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20.5 DNA Sequencing Is the Ultimate Way
to Characterize DNA Structure at the
Molecular Level
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Section 20.5
• The most common method of DNA sequencing is
dideoxynucleotide chain- termination sequencing
(Sanger sequencing) developed by Sanger
• This technique involves the addition of a small amount of
dideoxynucleotide, which causes DNA synthesis to
terminate
• Large-scale genome sequencing is automated and uses
fluorescent dye-labeled dideoxynucleotides
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Section 20.5
• Since the early 1990s, DNA sequencing has largely
been done through computer-automated Sanger
reaction-based technology (computer-automated highthroughput DNA sequencing)
– Generates large amounts of sequence DNA
– Enabled the rapid progress of the Human Genome Project
• Large-scale genome sequencing is automated and uses
fluorescent dye-labeled dideoxynucleotides
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Section 20.5
• The Sanger technique is outdated when it comes to
sequencing entire genomes
• Next-generation sequencing (NGS) technologies will
allow faster and cheaper genomic sequencing to take
place
• Through 2006, new sequencing technologies were
cutting sequencing costs in half about every two years
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next-generation sequencing
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