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2009 Application 8 Isolating, cloning Sequencing DNA
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OVERVIEW
Genomic DNA
Recombinant
plasmid vector
Plasmid
1. Cut out gene of interest and plasmid with the same restriction enzyme
2. leaving complementary sticky ends.
3. Mix the plasmid and genomic DNA fragments
4. and allow the sticky ends to anneal.
5. Add ligase to allow phosphodiester bonds to form.
6. Recombinant DNA molecules form in ligation mixture.
7. Transform competent* bacterial host cells in ligation mixture, which has
recombinant plasmid.
8. Need to identify bacteria host cell which has taken up recombinant plasmic
with gene of interest
(The genomic DNA sample will produce many restriction fragments).
Prepared by Nah S C
2009 Application 8 Isolating, cloning Sequencing DNA
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(a) Describe the natural function of restriction enzymes.
What are restriction enzymes?
o Produced by bacteria to cleave foreign DNA ( e.g. from bacteriophage ) into noninfective fragments
o recognise specific base sequences in double-stranded DNA and hydrolyse a
phosphodiester bond on each strand of the DNA at specific places known as restriction
sites.
o Most restriction sites are about 6 bases long and palindromic.
Features of restriction enzymes?
o Some restriction enzymes produce blunt ends while others produce sticky ends
(a) sticky ends  enzymes leave a staggered cut with single stranded ends.
These short extensions/ overhangs will anneal with complementary
single-stranded stretches on other DNA molecules cleaved with the same
restriction enzyme
(b) blunt ends  enzymes make a simple cut across both strands at a
single point
Why don’t they cut their own bacteria DNA?
o
(b)
Bacteria DNA protected by methylation –CH3 of adenine or cytosine within
restriction site.
Explain the formation of recombinant DNA molecule
1. ISOLATE vector (e.g. plasmid from bacteria) & gene of interest (e.g. DNA from
human cell)
2. Cut both DNA with the SAME RESTRICTION ENZYME.

Preferably one that produces complimentarty sticky ends
3. Mix plasmid and cut DNA.

Allows annealing of complimentary sticky ends between plasmid and gene of
interest
4. Add DNA ligase to seal fragments with phosphodiester bonds
5. a recombinant DNA (plasmid of bacteria) now contains a foreign (human) gene
Prepared by Nah S C
2009 Application 8 Isolating, cloning Sequencing DNA
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(c)(i) Outline the procedures for cloning a gene in a bacterial plasmid
(c) (ii) and describe the properties of plasmids that allow them to be used as DNA
cloning vectors.
What is a vector?
o is a DNA molecule into which fragments of DNA may be inserted.
o It then acts as an agent of transfer to carry the fragments of DNA into a host cell.
o Within the host, the vector replicates by using the DNA-synthesising machinery
(ie: DNA polymerase) of the host cell.
o Commonly used vectors include plasmids & phages/virus.
o Yeast have plasmids
What features must plasmids have to be useable in recombination
technology?
EcoRI
A selectable
marker gene
coding for
ampicillin (an
antibiotic)
resistance.
Insertion of gene of interest in either
ampr or tetr genes will inactivate
these genes. The region for such an
insertion is actually located within the
ampr or tetr gene.
BamHI
ampr
pBR322
tetr
A selectable marker gene
coding for tetracycline (an
antibiotic) resistance.
ori
Origin of replication, or ori.
Replication begins here with the formation of a
replication bubble. It allows the plasmid to
duplicate autonomously of bacterial chromosome.
(a) are capable of replicating independently of the bacterial chromosome
- so that during cloning, copies of the recombinant DNA molecule are passed
to daughter cells as the cell divides.
(b) Have high copy numbers
- present in the cell in multiple copies (30 – 40 per host cell) so that large
quantities of the recombinant DNA molecule can be obtained from each cell.
(c) have a single origin of replication
- host cell enzymes (e.g.: DNA polymerase) bind to initiate replication.
Prepared by Nah S C
2009 Application 8 Isolating, cloning Sequencing DNA
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(d) range in size (2 to several hundred kb).
- limits size of DNA fragment insterted
- Too large – hard to transform cells
(e) Contain 2 selectable markers
 Ampicillin + lacZ genes (coding for the enzyme -galactosidase).
 Ampicillin + tetracycline
 Has Restriction site within second gene

Gene of interest is inserted within the site inactivating the gene
(f) Gene of interest is insert under control of bacterial promoter (eukaryotic
promoter differ in sequence)
(c) (ii) Outline the procedures for cloning an eukaryotic gene in a bacterial
plasmid …………
(d) Explain how eukaryotic genes are cloned using E. coli cells to produce
eukaryotic proteins to avoid the problems associated with introns.
What is the problem faced with using eukaryotic gene for cloning into bacterial
cells?
1. bacteria lack post-transcriptional mechanism like mRNA splicing
a. introns are not removed
b. solution: use cDNA synthesized from mRNA by reverse transcriptase
c. chemically synthesised gene without its introns
2. bacteria RNA do not recognize eukaryote promoter
a. Need to place eukaryotic gene next to bacteria promoter.
3. Prokaryotes do not carry out post –translational modifications
a. E.g. no glycolysation
b. Mechanism for correct folding into 30 formation 40 structure
1. ISOLATE GENE OF INTEREST

mRNA for Anti-thrombin III is isolated from liver cells. (where it is found in high
quantities)

reverse transcriptase is used to make RNA-DNA hybrid

alkali used to partially break down RNA in hybrid

DNA polymerase used to synthesize complimentary DNA strand
2. Making RECOMBINANT PLASMID
Prepared by Nah S C
2009 Application 8 Isolating, cloning Sequencing DNA
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
cDNA is blunt ended

use terminal transferase to add cytosine residues to blunt ends thus forming
sticky ends

Cut plasmid to linearise leaving blunt ends

Use terminal transferase to as guanines to 3’ ends

Mix plasmid and gene of interest;

To allow annealing to each other

add ligase to seal nicks with phosphodiester bonds
Sticky ends
CCC
GGG
CCC
GGG
ampr
Inactivated tetr
Linearised
plasmid
ori
3. TRANSFORMATION of bacteria

transformation = process where a host cell assimilates external DNA

Treat with Ca2+ + heat shock 420C
- CaCl2 may be responsible for binding DNA to cell surface
What are the different types of plasmids that may result from the recombination?
o
Rejoined non-recombinant plasmids
o
Combination of 2 plasmids
o
Plasmid with several DNA fragments
o
Plasmid with gene of interest
4. SCREEN for bacteria cell with gene of interest
A. screen for cell that has taken up plasmid vector
o Plate bacteria on agar containing ampicillin
o Only cells with plasmid that have ampR gene produce resistance to ampicillin
and grow / other cells die
B How do we recognize cell clones that has taken up recombinant plasmids?
o By REPLICA PLATING
o Master plate with ampicillin
o Make a Replica plate of the master plate onto Petri dish with tetracycline
Prepared by Nah S C
2009 Application 8 Isolating, cloning Sequencing DNA
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o If gene of interest has been inserted in the tetracycline restriction site the
colony will not grow on the agar plate
o Colonies which grew on ampicillin but not later on tetracycline plates are the
successful recombinants.
Stamper
Sterile velvet
37C
12hr
Amp
Master plate (ampicillin)
Tet
Replica plate (tetracycline)
Tet
Replica plate (tetracycline)
Amp
Tet
Method 2: Use Plasmid carries 2 genes
o
AmpR  resistance to ampicillin
o
lacZ gene  encodes ß galactosidase
o hydrolyses sugar X-galactose (X-gal) to form blue product
o lacZ. Gene contains single restriction site recognize by the restriction
enzyme used
o Plate bacteria in ampicilin + X-gal.
o Colonies with functional lacZ. Gene. (no gene of interest inserted) produce ßgalactosidase, which hydrolyses Xgal and are blue
o Those with insert have a non-functional LacZ gene, and are unable to
metabolize Xgal: they therefore produce white colonies.
5. HOW DO YOU IDENTIFY RECOMBINANT CLONES WITH GENE OF INTEREST
a.
 Look for the gene
b.
look at/for its protein product
Nucleic acid probe hybridization
o Make a short single stranded nucleic acid (either RNA or DNA)
o Called DNA probe
o Complementary to gene of interest  with complementary base pair
o Probe labeled with radio active isotope or fluorescent tag
Prepared by Nah S C
2009 Application 8 Isolating, cloning Sequencing DNA
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Procedure
1. bacteria with recombinant plasmids are grown on a master plate
2. nitrocellulose filter is pressed on master plate
3. Filter is maked with X to note relative positions of colonies
4. filter treated with NaOH to lyse cells and denature DNA molecules to
single strands
5. wash away cell debris
6. bake to 80OC. ssDNA sticks to filter
7. incubate in radio active DNA probe
8. excess probe washed off
9. filter laid on photographic plate
10. black spots correspond to location where colonies carrying gene of
interest are found
6. LARGE SCALE PRODUCTION
 Colonies with gene of interest grown in bioreactor
 Culture induced to produce protein of interest
 Protein extracted, purified and packaged.
Prepared by Nah S C
2009 Application 8 Isolating, cloning Sequencing DNA
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What is a GENOMIC LIBRARY, What are their limitations?
1. Start with fragments of entire genome of an organism
2. each fragment cloned into vector (plasmid)
3. complete set of plasmids, each carrying a particular segment of the original
genome = genomic library
limitations
a. genomic DNA contain introns  cannot be sliced out if transformed into
bacterial cells
b. eukaryote genome too large  library too extensive to be screened
 may contain enormous tracts of non-coding DNA.
c. Gene of interest my be cut up by restriction enzyme  will not be a
single functional unit
Usefulness

Can be used to study introns, regulatory sequences (which would be
absent in cDNA library)
What is a cDNA library
Collection of cDNA clones generated in virto form mRNA sequences isolated forma
particular cell type/tissue.
Represents all of mRNA present in a particular tissue (total mRNA) which has been
converted back to dsDNA using reverse transcriptase
Use

Study coding sequence of gene (introns absent)

Want to trace changes in pattern of gene expression in same cell type at
different time in development of organism
Limitation

Genes not expresses at a certain stage of development of the tissue cannot be
harnessed
FEATURES
a. Lack introns  genes will be correctly expressed in bacteria
b. Cells have abundance of particular mRNA. Choice of tissues increases
chances of obtaining correct clone
(e)
Distinguish between a genomic DNA and cDNA library.
Point of
comparison
Genomic library
cDNA library
1.
Contains entire DNA content of
an organism including all coding
& non-coding sequences.
Contains entire protein-encoding
DNA content. Coding only
2.
Requires chromosomal DNA
isolation
Requires total mRNA isolation
Prepared by Nah S C
2009 Application 8 Isolating, cloning Sequencing DNA
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3.
Starting material can be from any
cell/tissue.
(total) mRNA should be isolated from
a cell/tissue where the particular
protein is likely to be produced in
large quantities.
4.
Genomic DNA is cleaved with
restriction enzymes before
cloning into a vector.
Messenger RNA reversetranscribed into cDNA before
cloning into a vector.
5.
Used for studying introns or
regulatory sequences associated
with a gene.
Cannot be used for studying introns
or regulatory sequences associated
with a gene.
OR
Used for studying the exact coding
sequence of the gene.
6.
Cannot be used for studying
physiological/developmental-based
changes in gene expression.
Used for tracing changes in
patterns of gene expression under
different
developmental/physiological
conditions.
7.
Can be used for the screening and
isolation of a gene, the expression
of which takes place in a cell that
is currently unknown.
Cannot be used for screening of a
gene when the cell type that
expresses it is unknown.
8. intactness
of genes
Genes may be fragmented since
restriction sites may be in middle of
gene sequence
Genes intact because source =
mRNA
9. frequency
of ‘genes’
Generally equal representation
Unequal proportions – some genes
represented in higher amounts
f. Outline 2 important proteins and other products that can be produced by
genetic engineering technique (eg. human growth hormone, anti-thrombin,
etc).
1
Outline how human growth hormone can be produced by genetic engineering
2
Outline how anti-thrombin can be produced by genetic engineering
3
Outline how other products can be produced by genetic engineering
1
Outline how human growth hormone can be produced by genetic engineering



Produced by cells of the anterior pituitary gland
Stimulates growth and cell reproduction at all stages of development
Excess leads to agromegaly (uncontrolled bone growth), deficiency leads to
dwarfism etc

mRNA was first obtained from pituitary gland to create a cDNA library.
Prepared by Nah S C
2009 Application 8 Isolating, cloning Sequencing DNA




2
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A small segment of the cDNA fragment was removed (codon 0 to 24). This
gap was replaced by a fragment, which provided the correct signals for
translation in E. coli.
Entire coding fragment is inserted behind E. coli lac promoter
The recombinant plasmid is used to transform a bacteria cell
Bacteria colony is induced to produce somatotrophin
Outline how anti-thrombin can be produced by genetic engineering
o Antithrombin is a small protein molecule that inactivates several enzymes of
the coagulation system.
o Is a glycoprotein produced by the liver
o Antithrombin deficiency is a rare hereditary disorder
o that generally comes to light when a patient suffers recurrent venous thrombosis
and pulmonary embolism.
o (Thrombosis is the formation of a clot or thrombus inside a blood vessel,
obstructing the flow of blood through the circulatory system.)
o (Pulmonary embolism is blockage of the pulmonary artery (or one of its
branches) by a blood clot, fat, air or clumped tumor cells.)
o mRNA was obtained from human liver to create a cDNA library.
o For efficient gene expression in E. coli, the E. coli trp promoter was used.
o
Prepared by Nah S C