Transcript DNA Technology

Introduction to Cloning and
Recombinant DNA Technology
David Bedwell, Ph.D.
Department of Microbiology
Office telephone: 934-6593
Email: [email protected]
The Powerpoint slides for
this lecture are available for download at:
http://www.microbio.uab.edu/bedwell/index4.html
Reference: Molecular Biology of the Cell, 5th Edition,
by Alberts et al., published by Garland Science, 2008.
Introduction to Cloning and Recombinant
Technology: Lecture Outline
•
•
•
•
•
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Introduction to Cloning and Recombinant
Technology: Lecture Outline
•
•
•
•
•
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
DNA is the genetic material of most
organisms (from bacteria to humans)
Plasmid
Chromosome: Most bacteria have one circular DNA chromosome ranging in size from
1,000 to 8,000 kilobase pairs.
Plasmid: Extrachromosomal genetic element also made of a circular DNA molecule.
Bacterial Genome: The collection of all of the genes present on the bacteria’s
chromosome or its extrachromosomal genetic elements.
Basics: Nucleotides are the
building blocks of DNA
Only in RNA,
not DNA
Deoxyribonucleic acid (DNA) is a long
double-stranded chain of nucleotides
• DNA is the hereditary material
passed on from generation to
generation.
• DNA is made up of four
nucleotides: A, C, G, and T.
• A always pairs with T.
• C always pairs with G.
• The two strands of DNA are in an
antiparallel configuration.
• Two complementary DNA strands
will separate when heated, and
will spontaneously pair together
again (hybridize) when cooled.
DNA Double Helix
Introduction to Cloning and Recombinant
Technology: Lecture Outline
•
•
•
•
•
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
What Does It Mean: “To Clone”?
Clone: a collection of molecules or cells, all identical to an
original molecule or cell
• To "clone a gene" is to make many copies of it - for
example, by replicating it in a culture of bacteria.
• Cloned gene can be a normal copy of a gene (= “wild
type”).
• Cloned gene can be an altered version of a gene (=
“mutant”).
• Recombinant DNA technology makes manipulating genes
possible.
Restriction Enzymes
• Bacteria have learned to "restrict" the possibility of
attack from foreign DNA by means of "restriction
enzymes”.
• Cut up “foreign” DNA that invades the cell.
• Type II and III restriction enzymes cleave DNA chains
at selected sites.
• Enzymes may recognize 4, 6 or more bases in
selecting sites for cleavage.
• An enzyme that recognizes a 6-base sequence is
called a "six-base cutter”.
Basics of type II Restriction Enzymes
• No ATP requirement.
• Recognition sites in double stranded DNA have a 2-fold
axis of symmetry – a “palindrome”.
• Cleavage can leave staggered or "sticky" ends or can
produce "blunt” ends.
Recognition/Cleavage Sites of Type II
Restriction Enzymes
Examples of Palindromes:
Cuts usually occurs at
a palindromic sequence
SmaI: produces blunt ends
5´ CCCGGG 3´
3´ GGGCCC 5´
EcoRI: produces sticky ends
5´ GAATTC 3´
3´ CTTAAG 5´
Don't nod
Dogma: I am God
Never odd or even
Too bad – I hid a boot
Rats live on no evil star
No trace; not one carton
Was it Eliot's toilet I saw?
Murder for a jar of red rum
Some men interpret nine memos
Campus Motto: Bottoms up, Mac
Go deliver a dare, vile dog!
Madam, in Eden I'm Adam
Oozy rat in a sanitary zoo
Ah, Satan sees Natasha
Lisa Bonet ate no basil
Do geese see God?
God saw I was dog
Dennis sinned
Type II restriction enzyme nomenclature
Why the funny names?
•
•
•
•
•
•
•
•
EcoRI –
BamHI –
DpnI –
HindIII –
BglII –
PstI –
Sau3AI –
KpnI –
Escherichia coli strain R, 1st enzyme
Bacillus amyloliquefaciens strain H, 1st enzyme
Diplococcus pneumoniae, 1st enzyme
Haemophilus influenzae, strain D, 3rd enzyme
Bacillus globigii, 2nd enzyme
Providencia stuartii 164, 1st enzyme
Staphylococcus aureus strain 3A, 1st enzyme
Klebsiella pneumoniae, 1st enzyme
Results of Type II Digestion
• Enzymes with staggered cuts  complementary ends
• HindIII - leaves 5´ overhangs (“sticky”)
5’ --AAGCTT-- 3’
5’ --A
3’ --TTCGAA-- 5’
3’ –TTCGA
AGCTT--3’
A--5’
• KpnI leaves 3´ overhangs (“sticky”)
5’--GGTACC-- 3’
5’ –GGTAC
3’--CCATGG-- 5’
3’ –C
C--
3’
CATGG--
5’
Results of Type II Digestion
• Enzymes that cut at same position on both strands
leave “blunt” ends
• SmaI
5’ --CCCGGG-3’ --GGGCCC--
3’
5’
5’
3’
--CCC
--GGG
GGG-CCC--
3’
5’
Restriction Endonucleases Cleave DNA
at specific DNA sequences
DNA Ligase joins DNA fragments together
• Enzymes that cut with staggered cuts result in
complementary ends that can be ligated together.
• HindIII - leaves 5’ overhangs (“sticky”)
5’ --A
3’ --TTCGA
AGCTT--3’
5’ --AAGCTT-- 3’
A--5’
3’ --TTCGAA-- 5’
• Sticky ends that are complementary (from digests with
the same or different enzymes) can be ligated together.
• Sticky ends that are not complementary cannot be
ligated together.
DNA Ligase can also join blunt ends
DNA fragments with blunt ends generated by different
enzymes can be ligated together (with lower efficiency),
but usually cannot be re-cut by either original restriction
enzyme.
• SmaI
• DraI
-CCC
-AAA
GGGTTT-
-CCCGGG-AAATTT-CCCTTT-AAAGGG-
• Ligations that re-constitute a SmaI or DraI site
(CCCGGG or AAATTT) can be re-cut by SmaI or DraI.
• Mixed ligation products (CCCTTT + AAAGGG) cannot be
re-cut by SmaI or DraI.
Any Complementary Ends Can be Ligated
• BamHI
-G
-CCTAG
GATCCG-
• BglII
-A
-TCTAG
GATCTA-
• Result
-GGATCT-CCTAGA-
No longer
palindromic, so not cut
by BamHI or BglII
Plasmids – vehicles for cloning
• Plasmids are naturally occurring
extrachromosomal DNA molecules.
• Plasmids are circular, double-stranded
DNA.
Ampr
Ori
pBR322
4361bp
Tetr
• Plasmids are the means by which
antibiotic resistance is often
transferred from one bacteria to
another.
LacZ
• Plasmids can be cleaved by restriction
enzymes, leaving sticky or blunt ends.
• Artificial plasmids can be constructed
by linking new DNA fragments to the
sticky ends of plasmid.
MCS
pUC18
Ori
Ampr
Cloning Vectors
Older cloning vector
• A cloning vector is a plasmid that can
be modified to carry new genes.
• Plasmids useful as cloning vectors must
have:
– An origin of replication.
– A selectable marker (antibiotic
resistance gene, such as ampr and
tetr).
– Multiple cloning site (MCS) (site
where insertion of foreign DNA will
not disrupt replication or inactivate
essential markers).
– Easy to purify away from host DNA.
Ampr
Ori
pBR322
4361bp
Tetr
Newer cloning vector
LacZ
MCS
pUC18
Ori
Ampr
Chimeric Plasmids
• Named for mythological beast
(chimera) with body parts from several
creatures.
• After cleavage of a plasmid with a
restriction enzyme, a foreign DNA
fragment can be inserted.
• Ends of the plasmid/fragment are
closed to form a "recombinant
plasmid”.
• Plasmid can replicate when placed in a
suitable bacterial host.
LacZ
MCS
pUC18-hCFTR
Ori
Ampr
DNA cloning requires restriction
endonuclease and DNA ligase
Consider a plasmid with a unique EcoRI site:
5' NNNNGAATTCNNNN 3'
3’ NNNNCTTAAGNNNN 5'
An EcoRI restriction fragment of foreign DNA
can be inserted into a plasmid having an EcoRI
cloning site by:
a) cutting the plasmid at this site with EcoRI,
b) annealing the linearized plasmid with the
EcoRI foreign DNA fragment, and,
c) sealing the nicks with DNA ligase.
5' NNNNGAATTCNNNN 3'
3' NNNNCTTAAGNNNN 5’
This results in a recombinant DNA molecule.
Introduction to Cloning and Recombinant
Technology: Lecture Outline
•
•
•
•
•
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Key features of DNA replication
are used in DNA sequencing
•
DNA synthesis occurs in the 5´ to 3´ direction.
•
DNA synthesis requires a template and a primer.
•
DNA replication is semi-conservative (one strand copied).
•
DNA replication is carried out by an enzyme called DNA
polymerase.
DNA synthesis requires a 3´-OH to make the next
phosphodiester bond during DNA synthesis
normal
dNTP
Dideoxy NTPs block DNA synthesis
H
ddNTPs block formation of the next
phosphodiester bond during DNA synthesis
A 3´-OH on the last ribose is needed for DNA synthesis
ddNTP
H
H
A nucleotidespecific stop in
DNA synthesis
A mixture of dNTPs and ddNTPs are
used in DNA sequencing
Polyacrylamide gel electrophoresis is used to
visualize the results of the sequencing reaction
Automated DNA sequencing with
fluorescent dyes coupled to each reaction
Fluorescent dye coupled to
reaction allows visualization
of di-deoxy termination
events by means of a laser
that detects the colored
product.
This shows four different
reactions as done with the
old manual sequencing.
Automated DNA sequencing output4 reactions carried out in one tube
Molecular Medicine: The Human Genome Project
3.2x109 nucleotide pairs
NCBI.nlm.nih.gov/genome/guide/human/index
Technology now exists to sequence everyone’s DNA
Took just 4 months,
$1.5 million to obtain
the entire DNA
sequence of James
Watson.
The genomes of many organisms have been sequenced
Genome resources for many
organisms are available
Introduction to Cloning and Recombinant
Technology: Lecture Outline
•
•
•
•
•
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Understanding the arrangement of genes
may help understand disease
Southern blot: One way to detect genome
structure and disease markers in genomic DNA
-Purify genomic DNA
-Digest with restriction enzyme
-Run agarose gel
Restriction fragment length polymorphisms
(RFLPs) can be associated with disease alleles
Southern Blot
Consider two alleles of a gene.
Allele A has 3 BamHI sites, while
allele a has only two BamHI sites.
probe
HpaI Digest
Variants
Nor3
mal 1 2
70% of carriers of the sickle cell
gene have a 13.0 kb HpaI fragment.
30% of carriers have 7.0 kb HpaI
fragment
Direct Detection of a Sickle Cell
Mutation by RFLP
A specific hemoglobin mutation
Wild Type
Pro Glu
Mutant
Pro Val
CCT GAG
DdeI site
CCT GTG
no DdeI site
[DdeI cuts at CTNAG]
AS AS SS AA
Gene encoding sickle cell b-subunit
Gene encoding Wild type b-subunit
Introduction to Cloning and Recombinant
Technology: Lecture Outline
•
•
•
•
•
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Polymerase Chain Reaction (PCR)
•
Allows quick identification of genetic markers:
Identify bacteria in infections
Identify viruses in virus infections
Paternity testing, genetic counseling, forensics
 Can exclude individuals, but cannot prove guilt.
•
Requires only small amounts of DNA.
•
A repetitive DNA synthesis reaction.
•
Thermostable DNA polymerase:
Isolated from bacteria in hot springs or near thermal vents
in the deep ocean.
•
Requires gene-specific DNA primers and
deoxyribonucleotide triphosphates (dNTPs).
Polymerase Chain Reaction (PCR)
A thermophilic (heat-loving) bacteria called Thermus aquaticus
is the source of Taq DNA polymerase used in PCR reactions.
The first round of PCR
94°C
37-65°C
70-75°C
PCR increases the yield of DNA exponentially
A typical PCR protocol

Begins with DNA containing a sequence to
be amplified and a pair of synthetic
oligonucleotide primers that flank the
sequence.

Next, denature the DNA to single strands
at 94˚C.

Rapidly cool the DNA (37-65˚C) and
anneal primers to complementary single
strand sequences flanking the target DNA.

Extend primers at 70-75˚C using a heatresistant DNA polymerase such as Taq
polymerase derived from Thermus
aquaticus.

Repeat the cycle of denaturing, annealing,
and extension 20-45 times to produce 1
million (220) to 35 trillion copies (245) of the
target DNA.

Extend the primers at 70-75˚C once more
to allow incomplete extension products in
the reaction mixture to extend completely.

Cool to 4˚C and store or use amplified
PCR product for analysis.
PCR cycle 28 – ~1 billion strands
5´
3´
5’
A
B
3´
5´
3´
A
A
B
B
3´
5’
2 original strands.
3´
28 strands starting with primer A, but with no
end.
5´
28 strands starting with primer B, but with no
end.
3´
~500,000,000 strands starting with primers A
(5´) and ending with primer B (referred to as
unit-length strand in previous figure).
5´
~500,000,000 strands starting with primer B
(5´) and ending with primer A (referred to as
unit-length strand in previous figure).
PCR in Medicine
• Since 1987, PCR has had a major impact on prenatal
diagnosis of single gene disorders.
• Also very important in carrier testing for genetic
diseases.
• Improved speed, accuracy and technical flexibility over
previous methods.
PCR and prenatal diagnosis
• For prenatal diagnosis, PCR used to amplify DNA from fetal cells
obtained from amniotic fluid.
• Single base changes then detected by one or more of following:
-dot blot (spot hybridization) with oligonucleotides specific for
known mutation.
-restriction enzyme analysis (RFLP).
-direct sequencing of DNA.
• Important to be certain of result so combination of two methods
provides confirmation.
• Many other conditions can be detected with same approach,
including:
-Tay-Sachs disease, phenylketonurea, cystic fibrosis, hemophilia,
Huntingdon's disease, Duchenne muscular dystrophy (DMD).
PCR to detect HIV
• PCR allows the direct detection of HIV genomes in patient
blood before the appearance of HIV antibodies.
• viral DNA/RNA only represents a minute proportion of total cell
DNA.
• Only a small fraction of blood cells are infected (1/10,000).
• also require high degree of specificity while targeting conserved
regions of DNA to guard against high level of genetic variability
characteristic of retroviruses.
• High risk of cross-contaminating sample with small amounts of
amplified DNA from previous sample requires extra precautions
to prevent false-positives.
• PCR can detect 10-20 copies of viral DNA from 150,000 human
cells.
PCR can be more rapid and accurate
than other diagnostic tests
• Diagnosis of the middle ear infection known as otitis media. The
technique has detected bacterial DNA in children's middle ear fluid,
signaling an active infection even when culture methods failed to
detect it.
• Lyme disease, the painful joint inflammation caused by bacteria
transmitted by tick bites, can be diagnosed by detecting the disease
organism's DNA contained in joint fluid.
• PCR is the most sensitive and specific test for Helicobacter pylori,
the disease organism now known to cause almost all stomach
ulcers.
• PCR can detect three different sexually transmitted disease
organisms on a single swab (herpes, papillomaviruses, and
chlamydia).
PCR in Forensics
Crucial forensic evidence may be present in very small quantities.
• often too little material for direct DNA analysis.
• but PCR can generate sufficient DNA from a single cell.
• PCR also possible on extensively degraded DNA.
• examples include DNA from single dried blood spot, saliva (on cigarette
butt), semen, tissue from under fingernails, hair roots.
Other advantages of PCR in forensic science are:
• relatively simple to perform and simple to standardize.
• results obtainable within 24 hours.
The major legal problems with PCR are the potential for crosscontamination between samples and the complexity of explaining what
the results mean to the jury.
PCR can exclude suspects
but cannot prove guilt
•
DNA typing is only one of many pieces of evidence that can lead to a
criminal conviction, but it has proved invaluable in demonstrating innocence.
•
Sometimes seemingly strong DNA evidence does not lead to a conviction
(see O.J. Simpson trial).
•
Dozens of cases have involved people who have spent years in jail for
crimes they did not commit until PCR exonerated them.
•
Even when evidence such as semen and blood stains are years old, PCR
can make unlimited copies of the tiny amounts of DNA remaining in the
stains for typing.
Variable Number of Tandem Repeat (VNTR)
analysis is commonly used in forensics
VNTR is based on hypervariable microsatellite sequence polymorphisms
within the human genome. These sequences (e.g., CACACA …) are found in
many locations in the human genome and vary greatly from person to person.
Using VNTR to compare forensic and
suspect samples
Individuals A & C are
excluded by this
analysis. The samples
from individual B will be
subjected to further tests.
Conclusions
•
•
•
•
•
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Questions?