Principles of Biochemistry Fourth Edition Chapter 23 Recombinant
DNA Technology Copyright 2006 Pearson Prentice Hall, Inc. Horton
Moran Scrimgeour Perry Rawn Slide 2 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 Chapter 23 - Recombinant DNA Technology Slide 3 Fig 23.1
Basic steps in the generation of a recombinant DNA molecule Slide 4
Fig 23.1 Basic steps in the generation of a recombinant DNA
molecule Slide 5 Fig 23.1 (cont) Slide 6 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) Slide 7 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 Slide 8 23.2 Cloning Vectors Cloning vectors can be:
plasmids, bacterio- phages, 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 Slide
9 Fig 23.2 Restriction enzymes can generate recombinant DNA Slide
10 A. Plasmid( ) 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 Slide 11 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 Slide 12 B. Bacteriophage Vectors
Efficient, commonly used vector for delivering DNA into a bacterial
cell Advantage over plasmid vectors is that transfection( ) is more
efficient than transformation( ) Disadvantage: DNA must be packaged
into phage particles in vitro Slide 13 Fig 23.4 Preparation and use
of phage vector Slide 14 Fig 23.4 Preparation and use of phage
vector Slide 15 Fig 23.4 (cont) (From previous slide) Slide 16 Fig
23.4 (cont) Slide 17 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 Slide 18 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 Slide 19 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 Slide 20 Fig 23.5 Yeast
artificial chromosome (YAC) Slide 21 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 Slide 22 A. Selection Strategies Use Marker Genes Bacterial
plasmid vectors can carry a -lactamase marker gene (marker genes
allow detection of cells) -Lactamase hydrolyzes -lactam antibiotics
(e.g. ampicillin) Only cells transformed with plasmids expressing
the -lactamase gene are ampicillin resistant and can grow in media
containing ampicillin (amp R ) Slide 23 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 (tet R ) can be inactivated by DNA
insertion making them tetracycline sensitive (tetS) Slide 24 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 Slide
25 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
-isopropylmalate dehydrogenase, (leucine biosynthesis pathway)
Cells transformed with a Leu2-containing plasmid can grow in the
absence of leucine Slide 26 Fig 23.6 Yeast shuttle vector is
propagated, selected in both E. coli and S. cerevisiae Slide 27 C.
Visual Markers: Insertional Inactivation of the -Galactosidase Gene
The lacZ gene of E. coli encodes -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 Slide 28 Fig 23. 7
Blue/white screen Blue colonies: cells transformed with cloning
vectors not containing inserts ( -galactosidase is active) White
colonies: cells transformed with recombinants. -Galactosidase gene
disrupted by insert Slide 29 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 Slide 30 Genomic Library Properties Genomic libraries
represent all the DNA from an organisms genome ( ) 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
Slide 31 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
Slide 32 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. Slide 33 Fig 23.8
Preparation of cDNA Slide 34 Fig 23.8 (continued) Slide 35
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
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) Slide 36 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 Slide
37 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) Slide 38 Fig 23.9 General procedure for screening DNA
library Slide 39 Fig 23.9 General procedure for screening DNA
library Slide 40 Fig 23.9 (cont) Hybridization Slide 41 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 Slide 42
Fig 23.4 Preparation and use of phage l vector Slide 43 Slide 44
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 Slide 45 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 Slide 46
Expression of a eukaryotic protein in E. coli Fig 23.11 Slide 47
Slide 48 Fig 23.11 (cont) Slide 49 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 Slide
50 Fig 23.12 Technique for creating a transgenic mouse Slide 51 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) Slide 52 larvaepupae adults eggs Transgenic oriental fruit
fly Slide 53 The Belgian Blue
http://bioethicsbytes.files.wordpress.com/2007/10/belgianblue1.jpg
Slide 54 Scaleless Chicken
http://bioethicsbytes.files.wordpress.com/2007/10/scaleslesschicken1.jpg
Slide 55 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 Slide 56 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) Slide 57
Slide 58 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 Slide 59 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 Slide 60
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 Slide 61 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)
Slide 62 Slide 63 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 Slide 64 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 Slide 65 Fig 19.34 DNA Fingerprinting Slide 66
Fingerprinting
(http://fig.cox.miami.edu/~cmallery/150/gene/c7.20.17.fingerprinting.jpg)
Slide 67 Fingerprinting
(http://www.scq.ubc.ca/wp-content/DNAfingerprintfamily.gif) Slide
68 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) Slide 69 Polymerase Chain Reaction
(PCR) Dr. Kary B. Mullis - the person who invented the polymerase
chain reaction (PCR) method in the early 1980s. Dr. Kary B. Mullis
earned the Nobel Prize for Chemistry in 1993. Slide 70 Slide 71
Slide 72 Slide 73 Slide 74 Identification of Codling Moth with
Specific DNA Markers (1) Slide 75 Identification of Codling Moth
with Specific DNA Markers (2) Slide 76 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 Slide 77 Fig 23.19 Oligonucleotide-
directed, site-specific mutagenesis Slide 78 Fig 23.19
Oligonucleotide- directed, site-specific mutagenesis Slide 79 Fig
23.19 (cont)
