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Horton • Moran • Scrimgeour • Perry • Rawn
Principles of Biochemistry
Fourth Edition
Chapter 23
Recombinant DNA Technology
Copyright © 2006 Pearson Prentice Hall, Inc.
Chapter 23 - Recombinant DNA Technology
23.1 Making Recombinant DNA
– Recombinant DNA molecules are constructed
with DNA from different sources
– Recombinant DNA molecules are created often
in nature
– Bacteriophage or eukaryotic virus infects a host
cell and integrates its DNA into the host
creating a recombinant DNA molecule
Fig 23.1
• Basic steps in the
generation of a
recombinant DNA
molecule
Fig 23.1
• Basic steps in the
generation of a
recombinant DNA
molecule
Fig 23.1 (cont)
Six Basic Steps in a Recombinant
DNA Experiment
1. Preparation of DNA. Vector and target DNA
2. Cleavage of DNA at particular sequences.
Insert DNA can be added at specific points
that have been cleaved
3. Ligation of DNA fragments. Joining of the
fragments
(continued)
Six Basic Steps (cont)
4. Introduction of recombinant DNA into
compatible host cells. Genetic transformation
is the uptake of foreign DNA by a host cell
5. Replication and expression of recombinant
DNA in host cells. Cloning vectors allow insert
DNA to be replicated in host cells
6. Identification of host cells that contain
recombinant DNA of interest. Screening a large
number of DNA clones for desired fragment
23.2 Cloning Vectors
• Cloning vectors can be: plasmids, bacteriophages, viruses, small artificial chromosomes
• Some vectors can be replicated autonomously
in a host cell, other vectors can be integrated
into the host chromosome
• Vectors have at least one unique cloning site:
a sequence cut by a restriction endonuclease to
allow site-specific insertion of foreign DNA
Fig 23.2
• Restriction enzymes
can generate
recombinant DNA
A. Plasmid(質體) Vectors
• Plasmids are small, circular DNA molecules
used as vectors for DNA fragments to 20 kb
• Replicate autonomously within a host cell
• Carry genes conferring antibiotic resistance,
used as marker genes for cells carrying vectors
• pBR322 was one of the first plasmid vectors
Fig 23.3 Plasmid vector pBR322
• pBR322 has 4361
base pairs
• Origin of replication
(ori)
• Antibiotic resistance
genes amp and tet
• Rop gene regulates
replication for ~20
copies of the
plasmid per cell
B. Bacteriophage l Vectors
• Efficient, commonly used vector for delivering
DNA into a bacterial cell
• Advantage over plasmid vectors is that
transfection(轉染作用) is more efficient than
transformation(轉形作用)
• Disadvantage: DNA must be packaged into
phage particles in vitro
Fig 23.4
• Preparation and
use of phage l
vector
Fig 23.4
• Preparation and
use of phage l
vector
Fig 23.4
(cont)
(From previous slide)
Fig 23.4 (cont)
Cosmids
• Cosmids combine the advantages of plasmid
and phage vectors
• Cosmids can accommodate large DNA
fragments and allow efficient transfection
• Recombinant DNA molecule can be
propagated as a plasmid in the host cell
C. Shuttle Vectors
• Shuttle vectors can replicate in either prokaryotic
or eukaryotic cells
• They can be used to transfer recombinant DNA
between prokaryotes and eukaryotes
• Useful for cloning eukaryotic DNA in bacteria,
and then expressing the gene products in a
eukaryotic cell
D. Yeast Artificial Chromosomes
as Vectors
• Large DNA fragments can be inserted
into artificial chromosomes that are
replicated in eukaryotic cells
• Such chromosomes must be linear and
contain a eukaryotic replication origin
• Yeast artificial chromosome (YAC) is a
shuttle vector
Fig 23.5 Yeast artificial chromosome (YAC)
23.3 Identification of Host Cells
Containing Recombinant DNA
• After a cloning vector and insert DNA have been
joined in vitro, recombinant DNA is introduced into
a host cell such as E. coli (transformation)
• Only a small percentage of cells take up the DNA
• Selection -cells are grown under conditions in
which only transformed cells survive
• Screening - transformed cells are tested for the
presence of the recombinant DNA
A. Selection Strategies Use Marker Genes
• Bacterial plasmid vectors can carry a
b-lactamase marker gene (marker genes allow
detection of cells)
• b-Lactamase hydrolyzes b-lactam antibiotics
(e.g. ampicillin)
• Only cells transformed with plasmids expressing
the b-lactamase gene are ampicillin resistant and
can grow in media containing ampicillin (ampR)
Selection or Screening by
Insertional Inactivation
• Insertional inactivation - insertion of a DNA
fragment within the coding region of a gene on a
vector results in inactivation of that gene
• If the gene product can be detected, this can be
used for selection and screening
• pBR322 gene for tetracycline resistance (tetR)
can be inactivated by DNA insertion making
them tetracycline sensitive (tetS)
Fig 23.3 Plasmid vector pBR322
• pBR322 has 4361
base pairs
• Origin of replication
(ori)
• Antibiotic resistance
genes amp and tet
• Rop gene regulates
replication for ~20
copies of the
plasmid per cell
B. Selection in Eukaryotes
• Yeast shuttle vectors may contain prokaryotic
genes for antibiotic resistance and yeast genes
for metabolite biosynthesis
• Yeast gene LEU2 encodes the enzyme
b-isopropylmalate dehydrogenase, (leucine
biosynthesis pathway)
• Cells transformed with a Leu2-containing
plasmid can grow in the absence of leucine
Fig 23.6
• Yeast shuttle
vector is
propagated,
selected in both
E. coli and S.
cerevisiae
C. Visual Markers: Insertional Inactivation
of the b-Galactosidase Gene
• The lacZ gene of E. coli encodes b-galactosidase
and cleavage of an artificial substrate produces a
blue dye (X-gal)
• Vectors without inserts in the lacZ gene give rise
to blue colonies in the presence of X-gal
• Vectors with DNA inserted in the lacZ gene do not
produce the enzyme and yields colonies which
are white
Fig 23. 7 Blue/white screen
• Blue colonies: cells
transformed with cloning
vectors not containing inserts
(b-galactosidase is active)
• White colonies: cells
transformed with recombinants.
b-Galactosidase gene disrupted
by insert
23.4 Genomic Libraries
• A method for isolating large quantities of
specific DNA fragments from organisms
• DNA library consists of all the recombinant
DNA molecules generated by ligating all the
fragments of a particular DNA into vectors
• Recombinant DNA molecules are then
introduced into cells for replication
Genomic Library Properties
• Genomic libraries represent all the DNA from
an organism’s genome (基因組)
• Partial (rather than total) restriction digestion is
used to ensure that every gene is represented
• Cosmid, YAC and BAC vectors used
• Genomic libraries include both expressed and
non-expressed DNA from the organism
23.5 cDNA Libraries Are Made
from Messenger RNA
• cDNA libraries represent all the mRNAs made
in a given cell or tissue
• cDNA (complementary DNA) is double-stranded
DNA made with reverse transcriptase
• Purification of mRNA relies on the polyA tails on
mature eukaryotic mRNA
• The more abundant rRNA and tRNA lack tails
Fig 23.8 Preparation of cDNA
RNase H: a ribonuclease that
cleaves the 3'-O-P-bond of RNA
in a DNA/RNA duplex to produce
3'-hydroxyl and 5'-phosphate
terminated products.
Fig 23.8 Preparation of cDNA
Fig 23.8 (continued)
Properties of cDNA libraries
• Using a cDNA library from a specific tissue
with abundant protein of interest increases
the chances of successfully cloning the gene
for that protein
• Specialized phage l vectors and plasmids
are used in constructing cDNA libraries
• cDNA libraries from mRNA do not include
introns or flanking sequences (much less
complex than genomic libraries)
23.6 Screening a Library
• Isolation of the desired recombinant DNA is
difficult (probability that a library of a given size
contains the particular clone of interest is):
P = 1 - (1-n)N
or
N = ln(1-P)/ln(1-n)
N = number of recombinant clones in library
P = probability of finding a particular clone
n = frequency of occurrence of the clone
Finding a Clone in a cDNA Library
• Probability of finding desired clone depends
on the abundance of the original DNA
molecule and not on the genome size
• n Represents the abundance of the relevant
mRNA molecule
• General procedure for screening a DNA
library with a probe (next slide)
Fig 23.9
• General procedure
for screening DNA
library
Fig 23.9
• General procedure
for screening DNA
library
Fig 23.9
(cont)
Hybridization
23.7 Chromosome Walking
• A recombinant DNA fragment from a nearby
region of the chromosome can be used as a
starting point for a chromosome walk to the
desired gene (Fig. 23.10, next slide)
• Overlapping DNA fragments are isolated in
successive screenings
• Larger and larger regions of DNA are cloned in
a “walk” along the chromosome
Fig 23.4
• Preparation and
use of phage l
vector
23.8 Expression of Proteins Using
Recombinant DNA Technology
• Cloned or amplified DNA can be purified and
sequenced or used to produce RNA and protein
• Such DNA can also be introduced into organisms
to change their phenotype
• Purification of proteins begins with
overproduction of the protein in a cell containing
the expression vector
A. Prokaryotic Expression Vectors
• Expression vectors - plasmids that have been
engineered to contain regulatory sequences for
transcription and translation
• Eukaryotic genes can be expressed in
prokaryotes
• Examples of sequences: strong promoters,
ribosome-binding sites, transcription terminators
Fig 23.11
• Expression of a
eukaryotic protein
in E. coli
Fig 23.11
Fig 23.11 (cont)
B. Expression of Proteins in Eukaryotes
• Prokaryotic cells may be unable to produce
functional eukaryotic genes
• Some expression vectors are for eukaryotes
• Recombinant DNA molecules can also be
integrated into the genomes of large
multicellular organisms
• Creates transgenic organisms
Fig 23.12
• Technique for creating
a transgenic mouse
Fig 23.13 Effect of an extra growth
hormone gene in mice
• Transgenic mouse
(left) carries a
gene for rat
growth hormone
• Normal mouse
(right)
Transgenic oriental fruit fly
eggs
larvae
pupae
adults
The Belgian Blue
http://bioethicsbytes.files.wordpress.com/2007/10/belgianblue1.jpg
Scaleless Chicken
http://bioethicsbytes.files.wordpress.com/2007/10/scaleslesschicken1.jpg
23.9 Applications of Recombinant
DNA Technology
• Somatic changes in tissues are not passed on
to subsequent generations
• Genome changes - germ cells are altered so
that changes are passed to descendents
• Agricultural genetic engineering: to produce
increased yield, resistance to insects, disease
or frost, altered ripening
• Introduction of nitrogen fixation into plants
A. Genetic Engineering of Plants
• Bacterial plasmid Ti can transform many kinds
of plants
• Transforming DNA (T-DNA) of the Ti plasmid
can be incorporated into E. coli-compatible
vector to yield plant-E. coli shuttle vectors
• Insertion of foreign DNA (firefly lucerferase) into
plants is shown on the next slide (Figure 23.14)
Fig 23.15 Insect-resistant tomato plants
• Insect-resistant
plant (left) contains
a gene that encodes
a protein that is
toxic to certain
insects that feed on
tomato plants
B. Genetic Engineering in Prokaryotes
Bacterial genomics research is important in:
• Combating bacterial resistance to antibiotics
• Studying how genome determines function
• Understanding microbial biochemistry and
pathology
• Developing drugs effective against pathogens
23.10 Applications to Human Diseases
• Proteins are now produced commercially from
cloned genes: insulin, interleukins, interferons,
growth hormones, coagulation factors etc
• Proteins are produced in bacteria, or transgenic
animals (blood, tissues, or milk)
• Mapping diseases to specific chromosomes
Restriction Fragment Length
Polymorphisms (RFLPs)
• RFLPs - genetic analysis based on variations
in length of genomic restriction fragments
• Patterns of disease inheritance can be traced
through a family
• RFLPs are detected by by incubating
fragmented DNA of many individuals with a
cloned DNA probe
• Hybridization pattern (Fig. 23.16 next slide)
RFLPs Studies
• The difference of even one nucleotide can
introduce or abolish a restriction site
• The pattern of hybridizing fragments can be
markedly changed
• Variations in a region of the genome near the
affected gene provide good screening
• Individuals can be screened for potential
diseases
RFLPs Uses in Forensic Medicine
• RFLP analysis can distinguish one person
from millions of others
• DNA samples from blood, hair, other tissues
• Restriction pattern is characteristic of each
individual (except identical twins)
• Comparisons of patterns can be used in
solving crimes
Fig 19.34
• DNA Fingerprinting
Fingerprinting
(http://fig.cox.miami.edu/~cmallery/150/gene/c7.20.17.fingerprinting.jpg)
Fingerprinting
(http://www.scq.ubc.ca/wp-content/DNAfingerprintfamily.gif)
23.11 The Polymerase Chain Reaction
Amplifies Selected DNA Sequences
• The polymerase chain reaction (PCR) is used
for amplifying a small amount of DNA
• Also can increase the proportion of a particular
DNA sequence in a mixed DNA population
• PCR technique is illustrated on the next 3 slides
(Figure 23.17, three cycles of the PCR reaction)
Polymerase Chain Reaction (PCR)
• Dr. Kary B. Mullis - the
person who invented the
polymerase chain reaction
(PCR) method in the early
1980’s.
• Dr. Kary B. Mullis earned
the Nobel Prize for
Chemistry in 1993.
Identification of Codling Moth with Specific
DNA Markers (1)
Identification of Codling Moth with Specific
DNA Markers (2)
23.12 Site-Directed Mutagenesis of Cloned DNA
• Powerful technique to introduce a desired
mutation directly into a gene
• Oligonucleotide is synthesized containing
mutation and flanking sequences of gene
• From oligonucleotide primer, DNA replication
produces a new copy of the mutated gene
• Important in structure-function studies of genes
and their protein products
Fig 23.19
• Oligonucleotidedirected,
site-specific
mutagenesis
Fig 23.19
• Oligonucleotidedirected,
site-specific
mutagenesis
Fig 23.19 (cont)