back to basics of pcr: comprehensive end-point pcr tips and tricks
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Sample to Insight
Back to basics: PCR – From set up to clean up 1
Back to basics: PCR – From set up to clean upMr. Abhishek Sharma, QIAGEN Discovery Sciences
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Sample to Insight
Legal disclaimer
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• QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention, or treatment of a disease.
• For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.QIAGEN.com or can be requested from QIAGEN
Technical Services or your local distributor.
Back to basics: PCR – From set up to clean up
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Agenda
Back to basics: PCR – From set up to clean up
PCR: Introduction
PCR primer design
PCR conditions
Enzymes used in PCR
PCR cycling
Agarose gel analysis
DNA cleanup
Overview of QIAGEN kits and products
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Agenda
Back to basics: PCR – From set up to clean up
PCR: Introduction
PCR primer design
PCR conditions
Enzymes used in PCR
PCR cycling
Agarose gel analysis
DNA cleanup
Overview of QIAGEN kits and products
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2
3
4
5
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PCR: Polymerase chain reaction
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Template DNA
Consideration
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PCR inhibitors
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Agenda
Back to basics: PCR – From set up to clean up
PCR: Introduction
PCR primer design
PCR conditions
Enzymes used in PCR
PCR cycling
Agarose gel analysis
DNA cleanup
Overview of QIAGEN kits and products
1
2
3
4
5
6
7
8
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PCR primer design
Guidelines for the design and use of primers
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Agenda
Back to basics: PCR – From set up to clean up
PCR: Introduction
PCR primer design
PCR conditions
Enzymes used in PCR
PCR cycling
Agarose gel analysis
DNA cleanup
Overview of QIAGEN kits and products
1
2
3
4
5
6
7
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PCR conditions
1. Primer annealing specificity and PCR buffers
2. A balanced combination of cations promotes
specific primer annealing
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3. Annealing temperatureQIAGEN tip: There is no need to calculate annealing temperature when using QIAGEN PCR kits. They work over a wide temperature range!
4. Magnesium ion concentrationThe Mg2+ concentration is generally higher than that of dNTPs and primers, and some optimization may be necessary for different template and primer concentrations.
5. PCR additivesCommonly used PCR additives include dimethyl sulfoxide(DMSO), bovine serum albumin (BSA) and glycerol.
6. Q-Solution
PCR conditions
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Guidelines for degenerate primer design and use
Primer sequence:• Avoid degeneracy in the 3 nucleotides at the 3’ end. If possible use Met- or Trp-
encoding triplets at the 3’ end
• To increase primer–template binding efficiency, reduce degeneracy by allowing some mismatches between the primer and template, especially towards the 5’ end, but not the 3’ end
• Try to design primers with less than 4-fold degeneracy at any given position
Primer concentration:• Begin PCR with a primer concentration of 0.2 μM
• In case of poor PCR efficiency, increase primer concentrations in increments of 0.25 μM until satisfactory results are obtained
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Agenda
Back to basics: PCR – From set up to clean up
PCR: Introduction
PCR primer design
PCR conditions
Enzymes used in PCR
PCR cycling
Agarose gel analysis
DNA cleanup
Overview of QIAGEN kits and products
1
2
3
4
5
6
7
8
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Enzymes used in PCR
• Most commonly used enzyme is Taq polymerase
• Hot-start DNA polymerase
• High-fidelity DNA polymerase
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Agenda
Back to basics: PCR – From set up to clean up
PCR: Introduction
PCR primer design
PCR conditions
Enzymes used in PCR
PCR cycling
Agarose gel analysis
DNA cleanup
Overview of QIAGEN kits and products
1
2
3
4
5
6
7
8
Sample to Insight
PCR cycling
In theory, each PCR cycle doubles the amount of amplicon in the reaction. Therefore, 10 cycles multiply the amplicon by a factor of ~1000 and so on.
Guidelines for determining the number of PCR cycles:
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Successful multiplex PCR and long-range PCR
Multiplex PCRMultiplex PCR employs different primer pairs in the
same reaction for simultaneous amplification of
multiple targets.
Factor MP: Supports macromolecule crowding
Amplification of long PCR productsOur LongRange PCR kits overcome the need
to develop specific long-range PCR protocols
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Agenda
Back to basics: PCR – From set up to clean up
PCR: Introduction
PCR primer design
PCR conditions
Enzymes used in PCR
PCR cycling
Agarose gel analysis
DNA cleanup
Overview of QIAGEN kits and products
1
2
3
4
5
6
7
8
Sample to Insight
Agarose gel analysis
Benefits• Enables quick and easy quantification of DNA
• Especially for small DNA fragments (such as PCR products)
• As little as 20 ng DNA can be detected by agarose gel electrophoresis with ethidium bromide staining
CoralLoad PCR Buffer• Directly load the PCR reaction onto an agarose gel
• Contains two marker dyes — an orange dye and a red dye This facilitate estimation of DNA migration distance and optimization of agarose gel run time
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Running an agarose gel
Agarose gel electrophoresis: Allows analysis of DNA fragments between 0.1 and 25 kb (e.g., genomic DNA digested with a frequently cutting restriction endonuclease).
Pulse-field gel electrophoresis enables analysis of DNA fragments up to 10,000 kb (e.g., undigested genomic DNA or genomic DNA digested with rare cutting restriction endonucleases).
A full tutorial on sample preparation is available on QIAGEN’s YouTube channel:
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Visual analysis of the gel
StainingTo allow visualization of the DNA samples, agarose gels are stained with an appropriate dye, such as ethidium bromide or SYBRR Green. SYBR Green is considered to be less hazardous, with respect to mutagenicity, than ethidium bromide.
• Addition of ethidium bromide prior to electrophoresis: Add ethidium bromide at a concentration of 0.5 μg/ml to the melted and subsequently cooled agarose, that is, just before pouring the gel. Mix the agarose–ethidium bromide solution well to avoid localized staining
• Addition of ethidium bromide after electrophoresis: Soak the gel in a 0.5 μg/ml solution of ethidium bromide (in water or electrophoresis buffer) for 30–40 minutes
Tips for handling ethidium bromide:
• Stock solutions of ethidium bromide (generally 10 mg/ml) should be stored at 4°C in a dark bottle or bottle wrapped in aluminum foil
• Rinse the gel with buffer or water before examining it to remove excess ethidium bromide. Staining buffer can be saved and re-used
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Agenda
Back to basics: PCR – From set up to clean up
PCR: Introduction
PCR primer design
PCR conditions
Enzymes used in PCR
PCR cycling
Agarose gel analysis
DNA cleanup
Overview of QIAGEN kits and products
1
2
3
4
5
6
7
8
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DNA cleanup
There are 3 main areas of DNA cleanup:
• Cleanup from enzymatic reactions, e.g., PCR• Nucleotide removal• Gel extraction and cleanup
The advantages of silica-membrane technology include:
• Yielding high-purity nucleic acids for use in most downstream applications
• Fast and inexpensive• No silica-slurry carry over, no alcohol precipitation
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Agenda
Back to basics: PCR – From set up to clean up
PCR: Introduction
PCR primer design
PCR conditions
Enzymes used in PCR
PCR cycling
Agarose gel analysis
DNA cleanup
Overview of QIAGEN kits and products
1
2
3
4
5
6
7
8
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Cleanup solutions from QIAGEN
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Cleanup kits overview
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End point PCR selection guide and cleanup selection guides
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Questions?
Thank you for attending
Contact QIAGEN Technical ServiceCall: 1-800-426-8157 for US
Call: +49 2103-29-12400 for EU
Email: [email protected]
Abhishek [email protected]
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