Download Polymerase Chain Reaction

Polymerase Chain Reaction
• Introduction
PCR:
> background
• Invented by Kary
Mullis in 1985
• Noble Prize 1993
• Extremely powerful
molecular biology
technique
DNA in the Cell
chromosome
cell nucleus
Double stranded
DNA molecule
Target Region for PCR
Individual
nucleotides
Practical applications of PCR
9
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9
9
9
9
9
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Amplify DNA for Cloning
Modify DNA for Cloning
Amplify DNA for sequencing without cloning
DNA sequencing reaction
Diagnose disease
Pathogen screening
Forensic analysis
Paternity/maternity (relatedness)
Population genetics (theoretical and
applied)
Polymerase Chain Reaction
• How does it work?
DNA Polymerization
• A few things that you know but should
keep in mind now.
Nucleotide 1
= one strand of DNA
Note polarity
Nucleotide 2
Nucleotide 3
Nucleotide 4
P
Nucleotide 1
S
T
one strand of DNA
P
Nucleotide 2
A
S
P
Nucleotide 3
C
S
P
Nucleotide 4
S
G
P
3’
S
T
S
A
Two strands of DNA
•Double-stranded DNA
•Anti-parallel
•Hydrogen bonds
•AT or GC pairs
P
P
A
T
S
S
P
P
C
G
P
C
3’
G
S
P
S
Polymerization of DNA
occurs from 5‘ to 3‘
P
S
S
5’
5’
DNA Polymerase Needs a Primer
ss DNA
3´
+
Nucleotides (dNTPs)
+
DNA polymerase
=
No DNA synthesis
5´
DNA Polymerase Needs a Primer
primer
ss DNA
5´
3´
3´
+
Nucleotides (dNTPs)
+
DNA polymerase
=
NoDNA
DNAsynthesis
synthesis
5´
DNA Polymerase Needs a Primer
primer
ss DNA
5´
3´
+
Nucleotides (dNTPs)
+
DNA polymerase
=
DNA synthesis
5´
DNA Polymerization
• How does nature do it?
Extension - The Replication Fork
5’
3’
3’
5’
3’
5’
5’
3’
5’
Primase
Single strand
binding
proteins
Laging Strand
Okazaki
fragment
5’
3’
5’
RNA
Primers
DNA
Polymerase
5’
3’
Helicase
Leading Strand
5’
3’
Functions And Their
Associated Enzymes
Function
• Melting DNA
Enzyme
Helicase
SSB Proteins
Topisomerase
• Polymerizing DNA DNA Polymerase
• Providing primer
Primase
• Joining nicks
Ligase
PCR
Purpose – Quickly make many copies of a region
of a DNA molecule
Method – Multiple rounds of DNA replication
Components in PCR reaction – Target DNA,
nucleotides, DNA polymerase, and primers
Temperature cycling – DNA replication
controlled by temperature…
Temperature Cycling in PCR
Temperature cycling – PCR process uses a
machine (thermocycler) in which PCR reaction
goes through ~30 cycles of three different
temperature changes:
~95ºC
– Melting temperature
50-65ºC
– Annealing temperature
72ºC
– Extension temperature
Your polymerase must tolerate 95ºC !
Hot water bacteria:
Thermus aquaticus
Taq DNA polymerase
Life at High Temperatures
by Thomas D. Brock
Biotechnology in Yellowstone
© 1994 Yellowstone Association for Natural Science
http://www.bact.wisc.edu/Bact303/b27
Polymerase chain reaction (PCR) analysis
1). primers are designed to flank the region to be amplified in target DNA
2). primers are annealed to denatured DNA
3). DNA is synthesized using Taq polymerase (from Thermus aquaticus)
4). primers are annealed again and the process is repeated through
20-30 cycles, geometrically amplifying the target sequence
5). DNA is analyzed by gel electrophoresis
1).
2).
3).
4).
Copying DNA - the polymerase chain reaction
Copying DNA - the polymerase chain reaction
Copying DNA - the polymerase chain reaction
Cells or tissue
RT-PCR
Reverse transcription
PCR
RNA isolation
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cDNA synthesis
RNA-dependent DNA polymerase (reverse transcriptase)
Gene specific priming
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Oligo(dT) priming
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Random priming
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PCR amplification
CONTAMINATION WITH GENOMIC DNA. DNAse treat RNA prior to experiment, use primers
either side of introns, and do control reaction WITHOUT reverse transcriptase.
Gene cloning
• Why would you want to clone a gene or cDNA?
– Sequence it
– Express it and make the encoded protein (bacteria, mammalian cells,
insect cells, yeast).
– Make knockout/transgenic constructs to generate GM mice.
• Popular methods for cloning PCR products include:
– classical cloning that uses restriction enzymes and ligase (REAL)
– TA Cloning®
• PCR primers can be used to modify the ends of PCR products.
– Add restriction sites.
– Add sequence encoding epitope tags for recognition by standard Abs.
Cloning your chosen gene
Plasmids are circular, independent genetic elements found in bacterial cells.
Put PCR product or DNA fragment into plasmid, and let bacteria do the amplification.
CONTAINS:
ORI – Origin of replication.
Selectable marker Ampicillin resistance
(Amp)
ORI
Amp
PCR product/DNA fragment
Transform
Into E.coli
ORI
Amp
Ampicillin containing plates
Grow at 37°c
In Ampicillin
Lyse cells and
Prepare DNA.
Simple cloning methods.
1. Cloning using restriction enzymes
– Design PCR primers with restriction enzyme (RE) sites
– Amplify DNA
– Digest with RE and ligate to vector digested with same enzyme
CTCTGGATCCAGATATG
AGAGACCTCGGTCTATAC
2. TA cloning
BamHI
CTCTG
GAGACCTAG
GATCCAGATATG
GTCTATAC
PCR
• Practical issues.
PCR COMPONENTS
• TWO PRIMERS complimentary to “flanking”
region of target
– Forward strand
– Reverse strand
• Nucleotides – “building blocks”
– A, T, C, G
• DNA Polymerase
– Taq (Thermus aquaticus)
Lots of pipetting required
PCR COMPONENTS
• Master Mix
– Primers
– Reaction components
– Dispensed into each tube for homogeneity
between samples
– Improves accuracy
– Limit need for pipetting small volumes
PCR CONTROLS
• NEGATIVE CONTROL
– PCR Master Mix ONLY
– NO Template DNA
• EXTRACTION BLANK
– Extraction Reagents ONLY
– NO Template DNA
• POSITIVE CONTROL
– Known positive sample
– Plasmid DNA
THERMAL CYCLER
Thermal Cycling Temperatures
95
Temperature
95oC
95oC
72oC
60oC
95oC
72oC
60oC
95oC
72oC
Typically 25-35 cycles
operformed during PCR
60 C
Single Cycle
30-40 cycles
REAL TIME PCR
USING SYBR GREEN
1
THE PROBLEM
• NEED TO QUANTITATE DIFFERENCES
IN mRNA EXPRESSION
• SMALL AMOUNTS OF mRNA
– SMALL AMOUNTS OF TISSUE
– PRIMARY CELLS
– PRECIOUS REAGENTS
– JUST LOW EXPRESSION OF YOU’RE
YOUR FAVROURITE GENE
2
THE PROBLEM
• QUANTITATION OF mRNA
–
–
–
–
northern blotting
ribonuclease protection assay
in situ hybridization
PCR
•
•
•
•
most sensitive
can discriminate closely related mRNAs
technically simple
but difficult to get truly quantitative results using
conventional PCR
3
NORTHERN
control
expt
target gene
internal control gene
actin, GAPDH, RPLP0 etc
Corrected fold increase = 10/2 = 5
Ratio target gene in experimental/control = fold change in target gene
fold change in reference gene
4
Standards/Reference genes
• same copy number in all cells
• expressed in all cells
• medium copy number advantageous
– correction more accurate
5
Standards
• The perfect standard does not exist
6
Standards
• Commonly used standards
– Glyceraldehyde-3-phosphate dehydrogenase
mRNA
– Beta-actin mRNA
– MHC I (major histocompatability complex I) mRNA
– Cyclophilin mRNA
– mRNAs for certain ribosomal proteins
• E.g. RPLP0
– 28S or 18S rRNA
7
REAL TIME PCR
• kinetic approach
• early stages
• while still linear
www.biorad.com
8
9
10
3. intensifier
1. halogen
tungsten lamp
2b. emission
filters
2a. excitation
filters
5. ccd
detector
350,000
pixels
4. sample plate
11
www.biorad.com
CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
AMOUNT OF DNA
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000
12
AMOUNT OF DNA
1600000000
1400000000
AMOUNT OF DNA
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000
1200000000
1000000000
800000000
600000000
400000000
200000000
0
0
5
10
15
20
25
30
35
PCR CYCLE NUMBER
A MOU N T OF D N A
CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
10000000000
1000000000
100000000
10000000
1000000
100000
10000
1000
100
10
1
0
5
10
15
20
25
PCR CYCLE NUMBER
30
35
13
AMOUNT OF DNA
0
5
10
15
20
25
30
35
PCR CYCLE NUMBER
14
A MOU N T OF D N A
0
5
10
15
20
25
30
35
PCR CYCLE NUMBER
15
Linear ~20 to ~1500
16
Linear ~20 to ~1500
17
SERIES OF 10-FOLD DILUTIONS
18
SERIES OF 10-FOLD DILUTIONS
19
threshold
Ct
SERIES OF 10-FOLD DILUTIONS
20
threshold = 300
21
threshold
22
An example
23
control
Δ Ct = target - ref
RPLP0 con
Δ Ct = 9.70
IL1-b con
av =19.93
experiment
av =29.63
Δ Ct = target - ref
IL1-b vit
Δ Ct = -1.7
RPLP0 vit
av =18.03
av =19.80
Difference = ΔCt-ΔCt
= ΔΔCt
= 9.70-(-1.7)
= 11.40
24
ΔΔCt = 11.40 for IL1-beta
• 2 ΔΔCt variant: assumes efficiency is
100% Fold change = 211.40 = 2702
25
The melting curve: -d(RFU)/dT
26
OVERVIEW
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results
27
OVERVIEW
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results
28
IMPORTANCE OF RNA
QUALITY
• Should be free of protein (absorbance
260nm/280nm)
• Should be undegraded (28S/18S ~2:1)
• Should be free of DNA (DNAse treat)
• Should be free of PCR inhibitors
– Purification methods
– Clean-up methods
29
OVERVIEW
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results
30
Importance of reverse
transcriptase primers
• Oligo (dt)
• Random hexamer (NNNNNN)
• Specific
31
REVERSE TRANSCRIPTION
• adds a bias to the results
• efficiency usually not known
32
OVERVIEW
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results
33
Importance of primers in PCR
•
•
•
•
specific
high efficiency
no primer-dimers
Ideally should not give a DNA signal
– cross exon/exon boundary
34
EXON 1
EXON 1
INTRON 2
EXON 2
EXON 2
DNA
RNA
35
How are you going to
measure the PCR product
• Directly
– Sybr green
– Quality of primers critical
• Indirectly
– In addition to primers, add a fluorescently
labeled hybridization probe
– Many different approaches to this, see
Bustin J.Mol.Endocrinol. (2000) 25:169
36
Importance of controls
• negative control (no DNA)
– checks reagents for contamination
• no reverse transcriptase control
– detects if signal from contaminating DNA
• positive control
– checks that reagents and primers work
– especially importance if trying to show
absence of expression of a gene
37
Standards
• same copy number in all cells
• expressed in all cells
• medium copy number advantageous
– correction more accurate
• reasonably large intron
• no pseudogene
• no alternate splicing in region you want
to PCR
38
SPECIAL THANKS TO
Christoph Schultes, who
• Set up qRT-PCR in our lab
• Made an overwhelming presentation on
it (you saw about 50% of it).
39
PCR trouble shooting
adapted from:
http://publish.uwcm.ac.uk/study/medicine/dip_biomed_meth/
website_pcr.ppt
CYCLING PARAMETERS
Denaturation;
93°C - 95°C
30 secs – 1min
Annealing;
37°C - 65°C
Touchdown?
30 secs – 1min
Extension;
72°C
1min
(+ 30secs per 500bp DNA)
25-35 cycles
Final extension
2-10mins
PCR
Agarose gel electrophoresis
3-4 hours
The final product
UV visualisation
ALWAYS REMEMBER!
PCR is a highly sensitive technique – contamination
with unwanted DNA can be a problem
Always run NEGATIVE controls
Include a positive control if appropriate
Use dedicated filtered tips and positive displacement
pipettes
Dedicated areas?
Can use UV cabinets
OPTIMISE PCR CONDITIONS AT THE OUTSET
The specific method should be ROBUST
Re-optimise for each set of primers
X
√
OPTIMISING PCR – THE REACTION COMPONENTS
• Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root,
saliva, semen
• Thermo-stable DNA polymerase
e.g. Taq polymerase
• Oligonucleotides
Design them well!
• Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
OPTIMISING PCR – THE REACTION COMPONENTS
• Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root,
saliva, semen
• Thermo-stable DNA polymerase
e.g. Taq polymerase
• Oligonucleotides
Design them well!
• Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
THE RAW MATERIAL
Tissue, cells, blood, hair root, saliva, semen
Obtain the best starting material you can.
Some can contain inhibitors of PCR, so they must be
removed e.g. Haem in blood
Good quality genomic DNA if possible
Blood – consider commercially available reagents
Qiagen– expense?
Empirically determine the amount to add
OPTIMISING PCR – THE REACTION COMPONENTS
• Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root,
saliva, semen
• Thermo-stable DNA polymerase
e.g. Taq polymerase
• Oligonucleotides
Design them well!
• Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
CHOOSE YOUR POLYMERASE WITH CARE
Number of options available
Taq polymerase
Pfu polymerase
others
•How big is the product?
100bp
40-50kb
•What is end purpose of PCR?
Sequencing - mutation detection
Need high fidelity polymerase
integral 3’
5' proofreading exonuclease activity
Cloning (TA cloning?)
TA CLONING OF PCR PRODUCTS REQUIRES As
A
A
PCR
product
Taq - yes
T
T
pGEM-T
pCR 2.1-TOPO
Pfu -
no
OPTIMISING PCR – THE REACTION COMPONENTS
• Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root,
saliva, semen
• Thermo-stable DNA polymerase
e.g. Taq polymerase
• Oligonucleotides
Design them well!
• Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
PRIMER DESIGN IS VITAL
•Length ~ 18-30nt (21nt)
•Base composition; 50 - 60% GC rich
pairs should have equivalent Tms
Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]
•Initial use Tm–5°C
•Avoid internal hairpin structures
no secondary structure
•Avoid A/T at the 3’ end
•Avoid overlapping 3’ ends – will form primer dimers
•Can modify 5’ ends to add restriction sites etc
OPTIMISING PCR – THE REACTION COMPONENTS
• Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root,
saliva, semen
• Thermo-stable DNA polymerase
e.g.Taq polymerase
• Oligonucleotides
Design them well!
• Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
TITRATE YOUR Mg2+ CONCENTRATION!
1
1.5
2
2.5
3
3.5
4 mM
Normally, 1.5mM MgCl2 is optimal
Best supplied as separate tube
Always vortex thawed MgCl2
Mg2+ concentration will be affected by the amount
of DNA, primers and nucleotides
USE MASTERMIXES WHERE POSSIBLE
Taken from http://info.med.yale.edu/genetics/ward/tavi/PCR.html
“ALL BLOCKS AND TUBES ARE EQUAL BUT
SOME ARE MORE EQUAL THAN OTHERS!”
G. Orwell (not!)
Taken from http://info.med.yale.edu/genetics/ward/tavi/PCR.html
ADDITIVES?
Depends on the PCR
Can be used where products are diffuse or absent
DMSO
Formamide
Glycerol
Stratagene - Perfect Match
http://taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html
THE PERFECT RESULT
Qiagen PCR
methods
If not………………………troubleshoot