Download Nucleic Acids Amplification and Sequencing

NUCLEIC ACIDS
Amplification and Sequencing
A. Extraction and Isolation of Nucleic Acids
• Cell lysis: rupture cell membrane and release
content
– treatment with hypotonic solution
– treatment with surfactants (SDS or Triton X-100)
– treatment with enzyme (lysozyme)
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CsCl density gradient centrifugation
• Separates DNA from RNA and Proteins
according to buoyant densities
1. Chromosomal DNA
2. Plasmid DNA
Total Cellular DNA Isolation
• Cell culture is transferred to detergent solution (buffer
containing SDS or Triton X-100)
• Detergent disrupts cell walls and DNA-protein complexes
• RNA is degraded with ribonuclease
• Proteins can be digested by a proteolytic enzyme
• DNA is extracted by precipitation with ethanol
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A protocol for Isolation of Plasmid DNA
• Centrifuge cell suspension
• Resuspend cell in cell lysis solution
• Mix then centrifuge lysate
cell debris are removed
• Transfer clear lysate to resin mini-column
• add ethanolic solution
– Proteins and RNA elute
• Elute DNA by washing the column with water that is Nuclease free
• Determine concentration by recording absorbance at 260 nm
– 1AU corresponds to 50µg/ml (50 ng/µL)
• Store DNA at -20ºC
RNA Isolation-The Proteinase K Method
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Problems
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RNA tends to form tight complexes with proteins
Ribonuleases are ubiquitous
Proteinase K Method
1. Cell lysis with hypotonic solution
2. Separation of DNA and debris by centrifugation
3. Dissociation of RNA-Protein Complexes
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Treatment with proteinase K
4. Separation of digestion products
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Extraction by phenol or chlorofrom
5. Precipitation of RNA with ethanol
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B. Nucleic Acid Amplification – The
Polymerase Chain Reaction (PCR)
• PCR is an in vitro technique for reproduction/
amplification of DNA sequences by enzyme catalysis
• Karl Mullis
– Discovered PCR in 1983
– Was awarded the Nobel Prize in 1993
• Applications
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Medical diagnosis
Genetics
Forensics
Clinical medicine
Principle of PCR - Reagents
• Excess of two primers: oligonucleotides that are
complementary to the ends of the targeted
nucleic acid region
• DNA polymerase: catalyses addition of
deoxynucleotide triphosphates (dNTPs) to
growing chain along a complementary chain
• Excess of the four deoxynucleotide
triphosphates (dATP, dCTP, dGTP and dTTP)
• Template DNA: DNA sample to be amplified
• Buffer containing Mg2+
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PCR Principle – Amplification Reaction
• Amplification reaction takes
place in a thermocycler
• Step 1- DNA denaturation – at
95ºC for 30 seconds
– H-bonds are disrupted and
double stranded DNA
(dsDNA) is denatured to
sinlge strand DNA (ssDNA)
– First cycle denaturation is
allowed during 2-5 minutes
• Step 2- Primer annealing: at 5060 ºC for 30 seconds
– Primers anneal with the
complementary region of the
target DNA
PCR Principle – Amplification Reaction
• Step 3- Primer extension (60
seconds)
– Synthesis of DNA is
catalyzed by DNA
polymerase at 72ºC
– Primer extension proceeds
from the 3’-end to the 5’end of the template
– 3’end of the hybridized
primer is extended along
the original DNA strand by
addition of nucleotides to
the chain
– Last extension step can be
extended (~5 minutes)
• 20 to 30 cycles are performed
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Example of a set of Experimental conditions
for PCR
• Concentration of
primer
– 50 ng/µL = 50 µg/mL
• Concentration of
dNTPS
– 10mM
PCR Principles - Results
• Theoretical yield
N m = N0 2n
N : number − of − molecules
n : number − of − cycles
• Limiting factors
¾ Depletion of reagents
¾ Amplification of longer strands during the first cycles
¾ Reaction efficiency:
• Empirical yield :
N m = N 0 (1 + x )n
x : reaction − efficiency(0 → 1)
• High specificity- only target sequence produced
• Fidelity – minimum polymerase induced errors
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Rate of Amplification During PCR
• Amplification of target section of DNA proceeds
according to three distinct phase.
– Starting phase• first cycle- copying from each end results in the synthesis of
two types of copy of longer length than target
• Second cycle- first copies of single stands target are
produced
• Third- first copies of double strands of the target
– Phase of exponential growth• Forth cycle: geometric increase starts
– A plateau
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Due to depletion of reagent
Deterioration of DNA polymerase
and inhibition of DNA polymerase by released phosphate
Hybridization of longer strands instead of hybridization of
ssDNA and primer
Rate of Amplification During PCR
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DNA Polymerase
• Must be heat stable
• Must have good polymerisation activity: i.e.
able to synthesize long stretches of DNA
• Pfu polymerase
– from hyperthermophilic archeon Pyrococcus
furiosis
– possesses 5’-3’ exonuclease activity which
improves fidelity of replication
– Incorrect nucleotides are recognized, removed
and replaced
Primers
• Short oligonucleotides complementary to the ends of the
target sequence
• Length: 10 – 30 nucleotides
• Two primers
– Forward primer
– Reverse primer
• Complementary sequence must be unique in the
template
• No intra of inter primer complementarity to prevent
formation of primer dimers
• Ideally equal number of each base
• Avoid long stretches of repetitive sequences
• Melting temperature for both primers should be similar a
should lie between 55 and 80°C
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dNTPs, Buffer, MgCl2, BSA
• Concentration of dNTPs should be equal
• Buffer pH, ionic strength adjusted to optimize the
catalytic efficiency of the polymerase used
• Ionic strength has a crucial influence on the specificity of
the PCR
– Typically 50 mM Tris-HCl, pH 8.3 with KCl of NaCl
• MgCl2: 0.5 and 5mM:
– Mg2+ forms a soluble complex with DNA and polymerase thus
bringing them in close proximity
– Mg2+ balances the charges on DNA.
– Mg2+ is a polymerase effector
– At low Mg2+ concentration activity of poly is decreased
– high concentration of Mg2+ increase annealing of the primers to
incorrect sites
• BSA: added to stabilize polymerase
Real-Time PCR
• Monitoring PCR product production
• Quantitative PCR (QT-PCR)
• DsDNA binding assay used to monitor product
– Dye is used that fluoresces upon binding to dsDNA
– Intercalator dyes
• Ethidium bromide
• Daunomycin
• Actinomycin
– Minor grooves binders : Distamycin, Netrospin, 4,6diamidino-2-phenylindole
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QT-PCR with Intercalator Dye
QT-PCR Probe-based Assay
• Rely on degradation of a target
fluorescent probe by 5’-3’
exonulease activity of the
polymerase
• Probe is a oligonucleotide
complementary to a region of the
target between two primers
• Fluorescent reporter is attached to
the probe at the 5’-end
• A quencher is attached to the 3’-end
• Probe is added to the PCR mixture
and hybridizes to the ssDNA after
denaturation
• Due to proximity of R and Q,
fluorescence is reduced
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QT-PCR Probe-based Assay
• During extension
reporter is cleaved
due to exonuclease
activity and fluoresces
• Fluorescence is
proportional to
number of cycles
Reverse Transcriptase-PCR (RT-PCR
• Used when RNA is available
– E.g. HIV virus
• The enzyme RNA-directed DNA polymerase ( reverse
transcriptase) is used
– Transcribes mRNA to its complementary strand of DNA
• RNA template is denatured at 72ºC
• RNA is cooled to 42ºC
– Primers anneal
• Reverse transcriptase catalyses the extension of primers
in the 5’ to 3’ direction generating cDNA
• Reaction is heated to 94ºC: reverse transcriptase is
inactivated
• PCR reagents are added for cDNA amplification
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C. Nucleic Acid Sequencing
• Chemical Cleavage Method
• Chain Terminator Method (Sanger or Dideoxy
Method)
• Walter Gilbert and Frederic Sanger were
awarded the Noble Prize in 1980 for their
pioneer work
C.1 Use of restriction enzyme in Sequencing
• Reduce native DNA to smaller fragments of approximately 800 base
pairs
– Restriction endonucleases recognize a specific base sequence of 4 to 8
bases within the dsDNA and cleave both strands at a point close to this
site
– BamHI (Bacillus amyloliquefacciens H) G♦GATCT
– XhoI (Xanthomonas holcicola) C♦TCGAG
– SalI (Streptomyces albus G) G♦TCGAC
– TaqI (Thermus aquaticus) T♦CGA (N6-Methlyladenine)
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Denature into ssDNA (melt dsDNA)
Separate ssDNA fragments by GE
Repeat digestion if there is DNA that is longer than 800 base pairs
Determine length of ssDNA products by comparing to standards
(restriction map)
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Note on Restriction Enzyme and Design of
DNA template
C.2 Chemical Cleavage (Maxam-Gilbert) Method
• ssDNA is labelled at
the 5’-end with a 32P
atom
– React ssDNA with [γ32P]ATP in the
presence of
polynucleotide kinase
– 5’-phosphate is
removed first with
alkaline phosphatase
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C.2 Chemical Cleavage/ Maxam-Gilbert Method)
• Labeled DNA is treated
with a reagent that cleaves
DNA at a particular type of
nucleotide
• Hydrazine cleaves DNA
before every C-nucleotide
at 1.5 M NaCl
• Reaction must have low
yield so as to obtain
random distribution of
different length due to
cuts at all the sites
• Labeled fragments are
separated by SDS-PAGE
Cleavage reaction for G residues
• When residues are
protonated, cleavage
occurs before both G
and A
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Example of cleavage before C
• Labeled DNA to be sequenced
– 32P-ACCTGACATCG
• Cleavage products
– 32P-ACCTGACAT
– 32P-ACCTGA
– 32P-AC
– 32P-A
Reagents for cleaving DNA
• Aliquot 1 Cleavage at only G
– DNA treated with Dimethyl sulfate (DMS)
– Methylation of G residues at the N7 position
– the glycoside bond of the methylated G residue is hydrolyzed and
the G residue is eliminated.
– Piperidine is added which reacts with hydrolyzed sugar residue,
cleavage of the backbone results
• Aliquot 2 cleavage at G and A
– Use acid instead of DMS
– Position of A revealed
• Aliquot 3: cleavage at C and T
– Treat with hydrazine, then piperidine
• Aliquot 4: cleavage at C only
– Treat with hydrazine in the presence of 1.5 M NaCl
– Position of T revealed
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Maxam Gilbert Method
C.3 The Chain Terminator Method (The Sanger or
Dideoxy)
• Synthesize complementary DNA like in PCR, but in the
presence of a chain terminating nucleotide
• Four aliquots each incubated with DNA polymerase, four
dNTPs and a suitable primer
• α-32P is incorporated in primer. This labels the
complementary strands for analysis
• A small amount of one of the 2’,3’-dideoxynucleotide
triphosphate (ddNTP) is added
– Incorporation of ddNTP terminates the reaction as there is no
free 3’-hydroxyl group
• Products are separated in parallel according to size by
GE and sequence is determined from the autoradiogram
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Sanger Method
Automated
Commercial Sanger
• Strategy I
– Four different reaction mixtures are set up
– Primers covalently bonded to fluorescing dye at the 5’-terminus
– Reaction products are separated by gel electrophoresis in
parallel lanes
– Laser induced fluorescence (LIF) is detected
• Strategy II
– Primers in each reaction mixture are labeled with different
fluorescent dyes
– Reaction mixtures are mixed at the end of the reaction and
separated in a single lane by gel electrophoresis
– The terminal base of each fragment is identified by the
fluorescence of the dye on the associated primer
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Commercial Sanger
• Strategy III
– A single vessel used
– Each of the ddNTPs is covalently bonded to a different
fluorescent dye
– The products are separated in single lane
– The terminal base is identified by the characteristic fluorescence
of the dye attached to the terminator
Examples of data expected when ddTTP and ddCTP are
the chain terminators
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Different fluorescent labels on each type of terminator
Univerisity of Michigan DNA Sequencing Core
Automated Sequencing Gel
http://seqcore.brcf.med.umichi.edu/doc/dnapr/sequencing.html
• Date from sequencer computer
– Colors detected in one lane (one sample)
– scanned from the smallest to the largest
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State-of-the-Art “Sanger Method” Instrument
High throughput
Computerized procedures
Robotics
Short analysis time afforded by CE
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