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Recombinant DNA

Technology

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings


Recombinant DNA Cloning Technology

genetic engineering – term for which a specific


definition is almost impossible

Is it:

any genetic manipulation that creates


change???
limited to biochemical intervention???
require creation of genetics that did not
previously exist???.

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings


Genetic Engineering

some possibilities

• isolation of individual genes


• production of proteins in large
quantities
• increased efficiency of production of
drugs
• creation of organisms with desirable
characteristics
• diagnosis of disease
• correction of genetic defects .
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• Genetic Engineering
• in livestock
• development of products for livestock
• Posilac – bovine growth hormone
used to increase milk production in
dairy cows
• direct improvement of livestock
• transgenesis, cloning

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• Genetic Engineering
• genetically modified organisms (GMOs)

• crops that have direct genetic


manipulations

• very controversial activity

• one of most common is

• crops with gene for resistance to


Round-Up herbicide .

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings


Manipulation of DNA

nature of DNA allows us to manipulate it


• application of heat breaks hydrogen
bonds
• application of heat does not break
phosphodiester bonds
therefore
• application of heat will separate strands of
duplex DNA
• application of heat will not separate
nucleotides .
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Cloning a DNA molecule
immediate goal of genetic engineering
insert a particular fragment of DNA into
another molecule
3 steps
• isolate DNA from an organism
• cut the DNA into pieces with
restriction enzymes and splice each
piece into a cloning vector
• introduce the recombinant DNA into
a host (such as E.coli, yeast or a cell)
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Steps in Genetic Engineering

1. Isolation of gene of interest


2. Isolation of plasmid DNA
3. Manipulation of DNA sequence
a. Cutting- Restriction enzymes
b. Splicing- DNA ligase
4. Transformation of bacteria
5. Selection of “correct” bacteria
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Cloning a DNA molecule

how to isolate a specific fragment?

use restriction enzymes (restriction


endonuclease)

cuts DNA at a restriction site .

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Cloning a DNA molecule
restriction site
short strand of DNA with specific base
sequence
frequently 4 to 6 bases
frequently symmetrical (palindrome)
eg. 5’ GAATTC 3’
3’ CTTAAG 5’
so will recognize in either direction .

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RESTRICTION ENDONUCLEASE
A restriction enzyme (or restriction endonuclease) is an enzyme
that cuts double-stranded DNA. The enzyme makes two incisions,
one through each of the sugar-phosphate backbones (i.e., each
strand) of the double helix without damaging the nitrogenous bases
 cleave the sugar-phosphate backbone of DNA

Thousands of restriction enzymes have been isolated from


bacteria, where they appear to serve a host-defense role.

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Restriction enzymes are classified biochemically into four types
(classes), designated Type I,Type II, Type III, and Type IV.

Type I and III, both the methylase and restriction


activities are carried out by a single large enzyme
complex. Both require ATP for their proper function.
In type II systems, the restriction enzyme is independent of
its methylase, and cleavage occurs at very specific sites that
are within or close to the recognition sequence. The vast
majority of known restriction enzymes are of type II, and it is
these that find the most use as laboratory tools. The first to be
discovered and utilized was EcoRI, which is staggered and
its recognition sequence is 5'-GAATTC-3'. Most type II
enzymes cut palindromic DNA sequences
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In type IV, the restriction enzymes target only
methylated DNA.
Restriction enzymes are named based on the bacteria in which
they are isolated in the following manner:
E : Escherichia (genus); co : coli (species); R : RY13 (strain); I :
First identified Order ID'd in bacterium
The substrates for restriction enzymes are specific sequences of
double-stranded DNA called recognition sequences.

The length of restriction recognition sites varies: The


enzymes EcoRI, SacI and SstI each recognize a 6 base-pair (bp)
sequence of DNA, whereas NotI recognizes a sequence 8 bp in
length, and the recognition site for Sau3AI is only 4 bp in
length.
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Different restriction enzymes which have the same recognition
site are called isoschizomers (SacI and SstI)

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Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Restriction recognitions sites can be unambiguous or
ambiguous: BamHI recognizes the sequence GGATCC
unambiguous. HinfI recognizes a 5 bp sequence starting with
GA, ending in TC, and having any base between (in the table, "N"
stands for any nucleotide)  ambiguous recognition site. XhoII
also ambiguous)

The recognition site for one enzyme may contain the restriction
site for another. The BamHI recognition site contains the
recognition site for Sau3AI.

most recognition sequences are palindromes - they read the


same forward (5' to 3' on the top strand) and backward (5' to 3'
on the bottom strand).

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Pattern of DNA Cutting by Restriction Endonuclease

1. 5' overhangs: The enzyme cuts asymmetrically within the


recognition site such that a short single-stranded segment extends
from the 5' ends. BamHI cuts in this manner.

The 5' or 3' overhangs generated by enzymes that cut


asymmetrically are called sticky ends or cohesive ends, because
they will readily stick or anneal with their partner by base
pairing.
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2. 3' overhangs: asymmetrical cutting within the recognition site,
the result is a single-stranded overhang from the two 3' ends. KpnI
cuts in this manner.

3. Blunts: Enzymes that cut at precisely opposite sites in the two


strands of DNA generate blunt ends without overhangs. SmaI is an
example of an enzyme that generates blunt ends.

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings


Use of restriction enzymes

restriction fragment
DNA fragment produced by a pair of
adjacent cuts in a DNA molecule

each restriction enzyme will produce a


characteristic set of fragments of varying
lengths

these fragments can be identified by size in


gel electrophoresis .

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings


Use of restriction enzymes

restriction enzymes have some common


characteristics

most recognize a single restriction site

restriction site recognized without regard


to the source of the DNA

number of cuts is determined by the


number of restriction sites .

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings


Use of restriction enzymes

Fragments can be manipulated


after identification by electrophoresis

can be inserted into bacteriophage,


plasmids or artificial chromosomes

called DNA cloning .

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Restriction site in DNA, showing symmetry of the
sequence around the center point

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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Cloning a DNA molecule
restriction fragments

many will spontaneously form circles

will straighten out if heated

will be permanently circular if DNA


ligase is applied (binds 3’ and 5’
ends) .

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings


Cloning a DNA molecule
if cuts are not opposite each other
one strand will extend belong the other
yields “sticky ends”
can adhere to other available sticky
ends since the bases would
complement
contrast to “blunt ends”
both strands cut at same place .

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Examples of how restriction enzymes cleave DNA

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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Cleavage of DNA by the restriction enzyme EcoRI

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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
What about different organisms??

restriction fragments of DNA from one


organism have the same sticky ends as
from another organism if they were
produced by the same restriction
enzyme .

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Recombinant DNA

DNA segment of interest is joined to a


vector (small DNA molecule that will
carry the DNA of interest)

recombinant molecule is introduced into a


cell by DNA transformation

when a transformation occurs we can say


that the DNA sequences have been
“cloned” .

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings


Vectors

important properties
have an “ori” - origin of DNA
replication
a dominant selectable marker – enables
cells that carry the plasmid to be
distinguished easily
unique restriction cleavage sites – sites
present just one in the vector for
insertion of the DNA fragments
.
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Vectors
common vectors
E coli plasmids
bacteriophages (virus that attacks
bacteria)
lambda ()
M13
cosmids
plasmid with cohesive ends of  phage

artificial chromosomes .
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Plasmids
Plasmid- small, circular,
extrachromosomal
DNA which replicates
independently of host
chromosomal DNA.
Most (experimental)
derived from a single
clinical specimen in
1974
Low copy # vs. high copy
number
Incompatible plasmids
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Isolation of Plasmid DNA

• Lysis by boiling
• Alkaline lysis
• Detergents or organic solvents

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Isolation of Plasmid DNA
Other Steps

• Centrifuge denatured proteins


• Precipitate nucleic acids with salt/EtOH
• Quantify

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Plasmid Map
Figure: Harpers Review of Biochemistry

• Ori
• antibiotic
resistance gene(s)
• restriction sites

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Insertion of a piece of DNA into the plasmid cloning
vector pUC19

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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
From E.Coli to a Map of Our Genes

• Research on E. coli revealed


that these bacteria have a
sexual mechanism that can
bring about the combining of
genes from two different cells
• This discovery led to the
development of recombinant DNA technology
– a set of techniques for combining genes from
different sources

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings


• DNA technology has many useful applications
– The Human Genome Project
– The production of vaccines, cancer drugs, and
pesticides
– Engineered
bacteria that
can clean up
toxic wastes

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BACTERIA AS TOOLS FOR MANIPULATING
DNA
In nature, bacteria can transfer DNA in three ways

• Transformation, the taking DNA enters


cell
up of DNA from the fluid
surrounding the cell
Fragment of
DNA from
another
bacterial cell

Bacterial chromosome
(DNA)

Figure 12.1A

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• Transduction, the • Conjugation, the union
transfer of bacterial of cells and the DNA
genes by a phage transfer between them
Mating bridge

Phage

Fragment of
DNA from Sex pili
another
bacterial cell
(former phage
host)

Donor cell Recipient cell


(“male”) (“female”)
Figure 12.1B Figure 12.1C

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• The transferred DNA is then integrated into the
recipient cell’s chromosome

Donated DNA Degraded DNA

Crossovers

Recipient cell’s Recombinant


chromosome chromosome

Figure 12.1D

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Bacterial plasmids can serve as carriers for gene
transfer
F factor (integrated)

• An F factor is a DNA Male (donor) cell

segment in bacteria that Origin of F replication

enables conjugation Bacterial chromosome

and contains an origin


F factor starts
replication and
transfer of chromosome

of replication Recipient cell

Only part of the


chromosome transfers

Figure 12.2A Recombination can occur


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• An F factor can exist as a
F factor (plasmid)

Male (donor)
cell
plasmid, a small circular
Bacterial
chromosome DNA molecule separate
F factor starts
from the bacterial
replication and
transfer chromosome

Plasmids

Plasmid completes
transfer and
circularizes

Cell now male


Figure 12.2B, C
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Plasmids are used to customize bacteria: An
overview

• Plasmids are key tools for DNA technology


– Researchers use plasmids to insert genes into
bacteria

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Bacterium 1 Plasmid Cell containing gene
of interest
isolated 2
DNA
isolated
3 Gene
Bacterial inserted
Plasmid into plasmid
chromosome
Gene of
Recombinant DNA interest DNA
(plasmid)

4 Plasmid put into


bacterial cell

Recombinant
bacterium

5 Cell multiplies with


gene of interest

Copies of gene Copies of protein

Gene for pest Clones of cell Protein used to


resistance make snow form
inserted into at higher
plants temperature

Gene used to alter bacteria Protein used to dissolve blood


for cleaning up toxic waste clots in heart attack therapy
Figure 12.3

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Enzymes are used to “cut and paste” DNA
Restriction enzyme

• Restriction enzymes
recognition sequence

1
cut DNA at specific
DNA

points
Restriction enzyme
cuts the DNA into
fragments

• DNA ligase “pastes” 2


Sticky end

the DNA fragments Addition of a DNA


fragment from
3

together
another source
Two (or more)
fragments stick
together by

• The result is 4
base-pairing

recombinant DNA
DNA ligase
pastes the strand

5
Figure 12.4 Recombinant DNA molecule
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Genes can be cloned in recombinant plasmids:
A closer look

• Bacteria take the recombinant plasmids and


reproduce
• This clones the plasmids and the genes they
carry
– Products of the gene can then be harvested

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• The potential uses of cloned genes fall into
two general categories.
• First, the goal may be to produce a protein
product.
– For example, bacteria carrying the gene for
human growth hormone can produce large
quantities of the hormone for treating stunted
growth.
• Alternatively, the goal may be to prepare
many copies of the gene itself.
– This may enable scientists to determine the
gene’s nucleotide sequence or provide an
organism with a new metabolic capability by
transferring a gene from another organism.

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E. coli
1 Isolate DNA Human cell
from two sources

2 Cut both
DNAs with
Plasmid the same
restriction DNA
enzyme
Gene V
Sticky ends

3 Mix the DNAs; they join


by base-pairing

4 Add DNA ligase


to bond the DNA covalently

Recombinant DNA
plasmid Gene V

5 Put plasmid into bacterium


by transformation

6 Clone the bacterium

Bacterial clone carrying many


copies of the human gene
Figure 12.5

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• The process of
cloning a
human gene in
a bacterial
plasmid can be
divided into
five steps.

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Cloned genes can be stored in genomic libraries

• Recombinant DNA Genome cut up


with restriction

technology allows
enzyme
Recombinant
plasmid Recombinant
the construction of OR
phage DNA

genomic libraries
– Genomic libraries are
sets of DNA fragments
containing all of an
Phage
Bacterial clone
clone

organism’s genes
Plasmid Phage

• Copies of DNA fragments


library library

can be stored in a cloned Figure 12.6

bacterial plasmid or phage


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Recombinant DNA libraries

genomic library – collection of clones


containing at least one copy of every
DNA sequence in the genome

chromosome libraries – collections of clone


of fragments of individual chromosomes

complementary DNA (cDNA) libraries –


collections of clones of DNA copies of
mRNA .

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Genomic libraries

at least one copy of every DNA sequence


how many clones needed?
yeast ~ 12000 kb in genome
need ~ 3600 recombinant DNA
molecules
humans ~ 3,000,000 kb in genome
need ~ 920,000 clones
must be large clones – use artificial
chromosomes .

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Chromosome library

make library of each chromosome

in humans – 24 chromosome libraries


22 autosomes, X, Y

can focus on individual chromosome if


gene of interest is already isolated on a
chromosome .

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Complementary DNA (cDNA)

genes in higher organisms are frequently


very large (several hundred kb)

size makes it difficult to clone

much of the length is in introns (are


removed during RNA processing)

would accomplish same thing if we could


copy info from processed RNA .

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Complementary DNA (cDNA)
cloning from mRNA
reverse transcriptase – enzyme required
to clone DNA from processed mRNA
molecule
product is called “complementary DNA”
or “cDNA”
DNA that codes for only the exon regions
of mRNA .

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The synthesis of complementary DNA (cDNA) from a
polyadenylated mRNA

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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Complementary DNA (cDNA)

can develop large quantity of cDNA using


PCR (polymerase chain reaction)

processed called

“reverse transcriptase PCR” .

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Screening for specific cDNA plasmids in a cDNA library
by using an antibody probe

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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Transformation of Bacteria

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CaCl2 Transformation

• Cells and DNA incubated together in CaCl2 at


0oC, then heat shock at 42oC
• How this makes cells “competent” to take up
DNA is not known
• Only a small percent of cells take up DNA-
must select for them

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Newer Methods of Transformation

• Lipofectin® and similar molecules


• Electroporation
• Microinjection

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Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Selection of “Correct” Bacteria

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Antibiotic Resistance Genes are a Part of Many
(Constructed) Plasmids

• Follow with replicate plating of transformants on


Amp and Tet
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Blue-White Screening
Promega Corp; Madison, WI

• pGEM-3Z, e.g.
– Ampr
– lacZ
– polycloning site in lacZ gene
– T7 promoter one side, SP6 other

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Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings