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QIAGEN Multiplex PCR Handbook

For fast and efficient multiplex PCR


Contents
Kit Contents
Shipping Conditions
Storage and Stability
Technical Assistance
Safety Information
Product Use Limitations
Product Warranty and Satisfaction Guarantee
Introduction

HotStarTaq DNA Polymerase
QIAGEN Multiplex PCR Buffer
Q-Solution
Product Description
Quality Control
Solutions and Reagents to Be Supplied by User
Protocols for Multiplex PCR
Standard Multiplex PCR Protocol
Multiplex PCR Protocol for Amplification of Microsatellite Loci
Multiplex PCR Protocol Using Q-Solution Troubleshooting Guide
Appendix
1. Starting template
2. General guidelines for multiplex PCR primer design
3. Handling and storage of primers
4. Number of PCR cycles
5. Separation of multiplex PCR products using agarose gel elect 4. Add template DNA (<= 1 g/50 l reaction)) to the individual PCR tubes containing the reaction mix.
For multiplex RT-PCR, the volume of cDNA added (from the RT reaction) as template should not exceed 10% of the final PCR volume. 5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 50 l mineral oil. 6. Program the thermal cycler according to the manufacturers instructions.
Optional: If a thermal cycler with a temperature gradient function can be used, determine the optimal annealing temperature by performing a gradient PCR. 7. Place the PCR tubes in the thermal cycler and start the cycling program as outlined in Table 3.
Each PCR program must start with an initial heat-activation step at 95C for 15 min to activate HotStarTaq DNA Polymerase.
After amplification, samples can be stored overnight at 28C or at 20C for longer storage. Table 3. Universal multiplex cycling protocol



* Tm determined according to the formula: Tm = 2C x (number of [A+T]) + 4C x (number of [G+C]).
For targets longer than 1.5 kb, an extension time of 2 min may improve performance. 8. Analyze samples using an appropriate detection system, for example agarose gel electrophoresis (see Ta ble 14 for choosing the optimal percentage of agarose), polyacrylamide gel electrophoresis, or capillary electrophoresis.
The optimal amount of PCR product to load, to give a satisfactory signal with your detection method should be determined individually.

Multiplex PCR Protocol for Amplification of Microsatellite Loci

Important notes before starting
  • Always start with the cycling conditions specified in this protocol.

  • If using an already established multiplex PCR system, use the previously established annealing temperature in combination with the cycling conditions specified in this protocol.

  • Annealing time must be 90 s.

  • Use equal concentrations (0.2 M) of all primers.

  • PCR must start with an activation step of 15 min at 95C to activate HotStarTaq DNA Polymerase (see step 7 of this protocol).

  • Optional: If a thermal cycler with a temperature gradient function can be used, determine the optimal annealing temperature by performing a gradient PCR.

Protocol

1. Thaw the 2x QIAGEN Multiplex PCR Master Mix (if stored at 20C), template DNA, distilled water, and primer mix. Mix the solutions completely before use.
It is important to mix the solutions completely before use to avoid localized concentrations of salts. Preparing a mixture of all primers avoids pipetting of individual primers for each experiment, reducing pipetting time and increasing reproducibility of results (for preparation of primer mix see Table 1) 2. Prepare a reaction mix according to Table 4.
The reaction mix typically contains all the components required for multiplex PCR except the template DNA. Prepare a volume of reaction mix 10% greater than that required for the total number of reactions to be performed. For reaction volumes less than 50 l, the 1:1 ratio of QIAGEN Multiplex PCR Master Mix to primer mix and template should be maintained as shown in Table 4.
Note: We strongly recommend starting with an initial Mg2+ concentration of 3 mM as provided by the 2x QIAGEN Multiplex PCR Master Mix. Table 4. Multiplex PCR components (reaction mix and template DNA)


* Provides a final concentration of 3 mM MgCl2.
A final primer concentration of 0.2 M is optimal for most primertemplate systems. However, in some cases using other primer concentrations (0.10.3 M) may further improve amplification performance.
For volumes less than 50 l, the 1:1 ratio of QIAGEN Multiplex PCR Master Mix to primer mix and template should be maintained. 3. Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tubes or plates.
Mix gently, for example by pipetting the reaction mix up and down a few times. Due to the hot start, it is not necessary to keep samples on ice during reaction setup. 4. A dd template DNA (<= 1 g/50 l reaction) to the individual PCR tubes containing the reaction mix.
For multiplex RT-PCR, the volume of cDNA added (from the RT reaction) as template should not exceed 10% of the final PCR volume. 5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 50 l mineral oil. 6. Program the thermal cycler according to the manufacturers instructions.
Optional: If a thermal cycler with a temperature gradient function can be used, determine the optimal annealing temperature by performing a gradient PCR. 7. Place the PCR tubes in the thermal cycler and start the cycling program as outlined in Table 5.
Each PCR program must start with an initial heat-activation step at 95C for 15 min to activate HotStarTaq DNA Polymerase.
After amplification, samples can be stored overnight at 28C or at 20C for longer storage. Table 5. Microsatellite cycling protocol

* Tm determined according to the formula: Tm = 2C x (number of [A+T]) + 4C x (number of [G+C]).
For targets longer than 0.5 kb, an extension time of 90 s may improve performance. 8. Analyze samples using an appropriate detection system, for example automatic gel-based DNA sequencers or those based on capillary electrophoresis.
The optimal amount of PCR product to load, to give a satisfac tory signal with your detection method should be determined individually.

Multiplex PCR Protocol Using Q-Solution

This protocol is designed for amplification of target sequences that do not work well under standard conditions and has been specifically optimized for multiplex PCR. Q-Solution changes the melting behavior of DNA and will often improve a suboptimal PCR caused by templates that have a high degree of secondary structure or that are GC-rich (>=65% GC content). It may also be helpful in other cases. When using Q-Solution for the first time, always perform parallel reactions with and without Q-Solution. This recommendation should also be followed if another PCR additive (such as DMSO) was previously used for a particular primer - template system.

We do not recommend combining GC-rich target sequences that either amplify poorly or not at all with target sequences that amplify easily in a multiplex PCR. Nevertheless, many of these primerCtemplate systems can be combined in a multiplex PCR using this protocol. When using Q-Solution, the following effects may be observed depending on the individual PCR assay:

Case A: Q-Solution enables amplification of a reaction that previously failed. Case B: Q-Solution increases PCR specificity in certain primertemplate systems. Case C: Q-Solution has no effect on PCR performance. Case D: causes reduced efficiency or failure of a previously successful amplification reaction. In this case, addition o f Q-Solution disturbs the previously optimal primertemplate annealing. Therefore, when using Q-Solution for the first time in a particular multiplex PCR assay, always perform reactions with and without Q-Solution.

Important notes before starting

  • Always start with the cycling conditions specified in this protocol.

  • If using an already established multiplex PCR system, use the previously established annealing temperature in combination with the cycling conditions specified in this protocol.

  • Annealing time must be 90 s.

  • Use equal concentrations (0.2 M) of all primers.

  • PCR must start with an activation step of 15 min at 95C to activate HotStarTaq DNA Polymerase (see step 7 of this protocol).

  • Optional: If a thermal cycler with a temperature gradient function can be used, determine the optimal annealing temperature by performing a gradient PCR.

Protocol

1. Thaw the 2x QIAGEN Multiplex PCR Master Mix (if stored at 20C), template DNA, distilled water, and primer mix. Mix the solutions completely before use.
It is important to mix the solutions completely before use to avoid localized concentrations of salts. Preparing a mixture of all primers avoids pipetting of individual primers for each experiment, reducing pipetting time and increasing reproducibility of results (for preparation of primer mix see Table 1).. 2. Prepare a reaction mix according to Table 6
The reaction mix typically contains all the components required for multiplex PCR except the template DNA. Prepare a volume of reaction mix 10% greater than that required for the total number of reactions to be performed. For reaction volumes less than 50 l, the 1:1 ratio of QIAGEN Multiplex PCR Master Mix to primer mix and template should be maintained as shown in Table 6.
Note: We strongly recommend starting with an initial Mg2+ concentration of 3 mM as provided by the 2x QIAGEN Multiplex PCR Master Mix. Table 6. Multiplex PCR components (reaction mix and template DNA)


* Provides a final concentration of 3 mM MgCl2.
A final primer concentration of 0.2 M is optimal for most primertemplate systems. However, in some cases using other primer concentrations (0.10.3 M) may further improve amplification performance.
For volumes less than 50 l, the 1:1 ratio of QIAGEN Multiplex Master Mix to primer mix, template, and Q-Solution should be maintained. 3. Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tubes or plates.
Mix gently, for example by pipetting the reaction mix up and down a few times.
Due to the hot start, it is not necessary to keep samples on ice during reaction setup. 4. Add template DNA (<= 1 g/50 l reaction) to the individual PCR tubes con taining the reaction mix.
For multiplex RT-PCR, the volume of cDNA added (from the RT reaction) as template should not exceed 10% of the final PCR volume. 5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 50 l mineral oil. 6. Program the thermal cycler according to the manufacturers instructions.
Optional: If a thermal cycler with a temperature gradient function can be used, determine the optimal annealing temperature by performing a gradient PCR. 7. Place the PCR tubes in the thermal cycler and start the cycling program as outlined in Table 7.
Each PCR program must start with an initial heat-activation step at 95C for 15 min to activate HotStarTaq DNA Polymerase.
After amplification, samples can be stored overnight at 28C or at 20C for longer storage. Table 7. Universal multiplex cycling protocol

* Tm determined according to the formula: Tm = 2C x (number of [A+T]) + 4C x (number of [G+C]).
For targets longer than 1.5 kb, an extension time of 2 min may further improve performance. 8. Analyze samples using an appropriate detection system, for example agarose gel electrophoresis (see Table 14 for choosing the optimal percentage of agarose), polyacrylamide gel electrophoresis, or capillary electrophoresis.
The optimal amount of PCR product to loa d, to give a satisfactory signal with your detection method should be determined individually.


Troubleshooting Guide

This troubleshooting guide may be helpful in solving any problems that may arise. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocol(s) in this handbook or molecular biology applications (see inside front cover for contact information).

Comments and suggestions
Little or no product 1. HotStarTaq DNA Polymerase
not activated Ensure that the cycling program included the HotStarTaq DNA Polymerase activation step (15 min at 95C) as described in step 7 of the protocols. 2. Pipetting error or missing reagent Repeat the PCR. Check the concentrations and storage conditions of reagents, including primers and template DNA. Mix all solutions before use. 3. Primer concentration not optimal Use a primer concentration of 0.2 M. For amplification of long targets (>= 1.5 kb), a primer concentration of 0.1 M may improve results. We do not recommend using primer concentrations higher than 0.3 - 0.4 M, as this may affect multiplex PCR fidelity. Check concentration of primer stock solutions. For calculation of primer concentration, refer to Appendix. 4. Insufficient number of cycles Increase number of PCR cycles. Refer to Appendix, for guidelines. 5. PCR cycling conditions not optimal Check that the correct cycling conditions were used (see Table 3, Table 5, and Table 7). Ensure that an annealing time of 90 s was used. If possible, perform a gradient PCR to determine the optimal annealing
temperature. 6. Primers degraded or of low quality Check functionality and specificity of primer pairs in single PCR reactions. Ensure that primers of sufficiently high quality were used. Check for possible degradation of the primers on a denaturing polyacrylamide gel. If necessary, make new dilutions of primer mix from primer stock solutions and store at 20C in small aliquots. Avoid repeated freeze/thaw cycles of the primer mix. 7. Annealing temperature too high Follow the recommendations given in Table 10 to determine the appropriate annealing temperature for your primers. Decrease annealing temperature in increments of 3C. Ensure that an annealing time of 90 s was used. If possible, perform a gradient PCR to determine the optimal annealing temperature. 8. GC-rich template or template
with a high degree of
secondary structure Using the same cycling conditions, repeat the multiplex PCR using 0.5x Q-Solution. Templates with a very high GC content that do not amplify under these conditions should rophoresis
6. Sensitive multiplex PCR assays
7. Guidelines for special multiplex PCR applications
8. Optimization of reaction conditions for special multiplex PCR applications Kit Contents


Shipping Conditions

The QIAGEN Multiplex PCR Kit is shipped on dry ice, but retains full activity at room temperature (1525C) for 2 days.

Storage and Stability

The QIAGEN Multiplex PCR Kit, including Q-Solution and distilled water, should be stored immediately upon receipt at 20C in a constant-temperature freezer. When stored under these conditions and handled correctly, these products can be kept at least until the expiration date (see the inside of the kit lid) without showing any reduction in performance. The QIAGEN Multiplex PCR Master Mix can be stored at 28C for up to 6 months.

Technical Assistance

At QIAGEN we pride ourselves on the quality and availability of our technical support. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products. If you have any questions or experience any difficulties regarding the QIAGEN Multiplex be combined in a separate multiplex PCR assay using 1x Q-Solution. 9. Insufficient starting template Increase amount of starting template up to 1 g per 50 l reaction. 10. Primer design not optimal Review primer design. Refer to Appendix, for general guidelines on multiplex PCR primer design. 11. Problems with starting template Check the concentration, storage conditions, and quality of the starting template (see Appendix). If necessary, make new serial dilutions of template nucleic acid from stock solutions. Repeat the multiplex PCR using the new dilutions. 12. PCR product too long The optimized protocols allow amplification of target sequences up to 1.5 kb. We recommend using an extension time of 2 min for target sequences between 1.52.0 kb. Increase the extension time in increments of 30 s for each additional 0.5 kb of target sequence. 13. PCR overlaid with mineral oil
when using a thermal cycler
with a heated lid When performing PCR in a thermal cycler with a heated lid, do not overlay the PCR samples with mineral oil if the heated lid is
switched on. This may decrease the yield of PCR product. 14. Problems with the thermal cycler Check the power to the thermal cycler and that the thermal cycler has been correctly programmed. Not all products are detectable, or some products are barely detec table 1. Primers degraded or of low quality Check functionality and specificity of primer pairs in single PCR reactions. Ensure that primers of sufficiently high quality were used. Check for possible degradation of the primers on a denaturing polyacrylamide gel. If necessary, make new dilutions of primer mix from primer stock solutions and store at 20C in small aliquots. Avoid repeated freeze/thaw cycles of the primer mix. 2. Primer concentration not optimal Use a primer concentration of 0.2 M. For amplification of long targets (>=1.5 kb), a primer concentration of 0.1 M may improve results. We do not recommend using primer concentrations higher than 0.3 - 0.4 M, as this may affect multiplex PCR fidelity. Check concentration of primer stock solutions. For calculation of primer concentration, refer to Appendix. 3. PCR cycling conditions not optimal Check that the correct cycling conditions were used (see Table 3, Table 5, and Table 7). Ensure that an annealing time of 90 s was used. If possible, perform a gradient PCR to determine the optimal annealing temperature. 4. No final extension step, or final
extension step was not optimal Ensure that the final extension step was performed as described in Table 3, Table 5, and Table 7. When detecting multiplex PCR products under native conditions, a final extension step of 15 min at 68C for multiplex systems with more than 10 PCR products, or for PCR products longer than 1.5 kb may improve results. For microsatellite analysis, a final extension step of 30 min at 60C should be used. 5. Annealing temperature too high Follow the recommendations given in Table 10 to determine the appropriate annealing temperature for your primers. Decrease annealing temperature in increments of 3C. Ensure that an annealing time of 90 s was used. If possible, perform a gradient PCR to determine the optimal annealing temperature. 6. GC-rich template or template
with a high degree of
secondary structure Using the same cycling conditions, repeat the multiplex PCR using Q-Solution.Templates with a very high GC content that do not amplify under these conditions should be combined in a separate multiplex PCR assay using 1x Q-Solution. 7. Sensitivity not high enough If your assay requires very high sensitivity, the sensitivity of the multiplex PCR can be further increased by a prolonged annealing time of 3 min. Additional products detectable 1. PCR cycling conditions not optimal Check that the correct cycling conditions were used (see Table 3, Table 5, and Table 7). Ensure that an annealing time of 90 s was used. If possible, perform a gradient PCR to determine the optimal annealing temperature. 2. Too many PCR cycles Too many PCR cycles may increase nonspecific background. Determine the optimal number of cycles by decreasing the number of PCR cycles in increments of 3 cycles. 3. Annealing temperature too low Follow the recommendations given in Table 10 to determine the appropriate annealing temperature for your primers. Increase annealing temperature in increments of 2C. Ensure that an annealing time of 90 s was used. If possible, perform a gradient PCR to determine the optimal annealing temperature. 4. Mg2 concentration not optimal Use an initial Mg2 concentration of 3 mM as provided by the QIAGEN Multiplex PCR Master Mix. In rare cases, an increase in Mg2 concentration may increase product yield. Perform multiplex PCR with different final concentrations of Mg2 by titrating in 0.5 mM steps. 5. Primer concentration not optimal Use a primer concentration of 0.2 M. For amplification of long targets (>= 1.5 kb), a primer concentration of 0.1 M may improve results. We do not recommend using primer concentrations higher than 0.3 - 0..4 M, as this may affect multiplex PCR fidelity. Check concentration of primer stock solutions. For calculation of primer concentration, refer to Appendix. 6. Primer design not optimal Review primer design. Refer to Appendix, , for general guidelines on multiplex PCR primer design. 7. Some primers generate more
than one specific product Multiplex primer pairs bind in close proximity to each other, for example during amplification of multiple parts of a genomic locus. Additional larger products may be generated by outside primers, refer to Appendix. 8. Primers degraded or of low quality Check functionality and specificity of primer pairs in single reactions. Ensure that primers of sufficiently high quality were used. Check for possible degradation of the primers on a denaturing polyacrylamide gel. If necessary, make new dilutions of primer mix from primer stock solutions and store at 20C in small aliquots. Avoid repeated freeze/thaw cycles of the primer mix. 9. Amplification of
pseudogene sequences Primers may anneal to pseudogene sequences and additional PCR products may be amplified. Review primer design to avoid detection of pseudogenes. Refer to Appendix for general guidelines on multiplex PCR primer design. 10. No final extension step, or final
extension step was not optimal Ensure that the final extension step was performed as described in Table 3, Table 5, and Table 7. When detecting multiplex PCR products under native conditions, a final extension step of 15 min at 68C for multiplex systems with more than 10 PCR products, or for PCR products longer than 1.5 kb may improve results. For microsatellite analysis, a final extension step of 30 min at 60C should be used. 11. GC-rich template or template
with a high degree of
secondary structure Using the same cycling conditions, repeat the multiplex PCR using Q-Solution. Templates with a very high GC content that do not amplify under these conditions should be combined in a separate multiplex PCR assay using 1x Q-Solution. If detecting multiplex PCR products under non-denaturing conditions (e.g., on agarose gels or native polyacrylamide gels): Some products are smeared, or additional products are observed 1. Too many PCR cycles Too many PCR cycles may increase nonspecific background. Determine the optimal number of cycles by decreasing the number of PCR cycles in increments of 3 cycles. 2. Too much starting template Check the concentration of the starting template DNA. Repeat the multiplex PCR using less DNA (i.e., <= 1 g per 50 l reaction). 3. No final extension step, or final
extension step was not optimal Ensure that the final extension step was performed as described in Table 3, Table 5, and Table 7. When detecting multiplex PCR products under native conditions, a final extension step of 15 min at 68C for multiplex systems with more than 10 PCR products, or for PCR products longer than 1.5 kb may improve results. For microsatellite analysis, a final extension step of 30 min at 60C should be used. 4. Incomplete renaturation of PCR
products due to either low GC
content or long length of
PCR products Use a final extension step of 15 min at 68C. We recommend this for multiplex systems with more than 10 PCR products, or for PCR products l onger than 1.5 kb. 5. Double-stranded products
melt during electrophoresis PCR products with a low GC content may melt if electrophoresed at high voltages. Reduce the voltage to prevent the running buffer overheating. 6. Background using silver staining Purify PCR products before loading onto the gel (e.g., using the QIAquick Gel Extraction Kit or MinElute Gel Extraction Kit. If detecting fluorescently labeled multiplex PCR products under denaturing conditions (e.g., on an automatic sequencer, or by capillary electrophoresis): Additional products are observed 1. Loading volume is too high Loading of large volumes may result in additional peaks. Decrease the loading volume until the background is decreased to a satisfactory level with acceptable peak heights (typically peak heights <2000 relative fluorescent units on ABI PRISM 310 or 377 Genetic Analyzer). 2. Faint peaks (stutter peaks)
before main peak Amplification of some microsatellite DNA sequences may lead to artifacts, referred to as stutter peaks, which are usually one repeat unit shorter than the main peak. We recommend decreasing the loading volume as described above. If the length of the faint peak is one base shorter than the main peak, refer to n1 products detected. 3. Sample not completely denatured Denature the samples before loading by heating. 4. n1 products detected Ensure that the final extension step was performed as described in Table 3, Table 5, and Table 7. If the final extension step was correctly performed, decrease the number of cycles and/or template amount. 5. Differing signal intensities
from fluorescent dyes Different fluorescent dyes may give differing signal intensities on a particular detection instrument, although comparable amounts of PCR product are generated. We recommend combining fluorescent dyes for multiplex PCR according to the instructions of the detection instruments manufacturer. Faint peaks or no allele peaks 1. Poor capillary electrophoresis
injection (size standard also affected) Inject the sample again. Check the syringe O-ring for leakage. Check that the fluores) cence detection instrument is functioning correctly. 2. Poor quality formamide used Use high-quality formamide for the analysis of samples on the ABI PRISM 310 Genetic Analyzer. The conductivity of the formamide should be <100 S/cm. If performing RT-PCR: Little or no product RT reaction error On average only 1030% of the starting RNA in the RT reaction is transcribed into cDNA. The volume of the RT reaction added as template should not exceed 1 0% of the final PCR volume. Additional larger products Contamination with genomic DNA Additional larger products may result from amplification of contaminating genomic DNA. Pretreat RNA with DNase I (e.g., using the QIAGEN RNase-Free DNase Set). Alternatively, use primers located at splice junctions of the target mRNA to avoid amplification from genomic DNA.

1. Starting template

Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification.

Quality of starting template

Since PCR consists of multiple rounds of enzymatic reactions, it is more sensitive to impurities such as proteins, phenol/chloroform, salts, ethanol, EDTA, and other chemical solvents than single-step enzyme-catalyzed processes. QIAGEN offers a complete range of nucleic acid preparation systems, ensuring the highest-quality templates for PCR. This includes the QIAprep system for rapid plasmid purification, the QIAamp and DNeasy systems for rapid purification of genomic DNA and viral nucleic acids, and the RNeasy system for RNA preparation from a variety of sources. For more information about QIAprep, QIAamp, DNeasy, PAXgene Blood DNA System, and RNeasy products, contact one of our Technical Service Departments (see inside front cover).

Quantity of starting template

The annealing efficiency of primers to the template is an important factor in PCR. Owing to the thermodynamic nature of the reaction, the primer:template ratio strongly influences the specificity and efficiency of PCR and should be optimized empirically. If too little template is used, primers may not be able to find their complementary sequences. Too much template may lead to an increase in mispriming events. As an initial guide, spectrophotometric and molar conversion values for different nucleic acid templates are listed in Tables 8 and 9, respectively.

Table 8. Spectrophotometric conversions for nucleic acid templates


Table 9. Molar conversions for nucleic acid templates



* Base pairs in haploid genome
For single-copy genes

2. General guidelines for multiplex PCR primer design

Prerequisites for successful multiplex PCR include the design of optimal primer pairs, the use of appropriate primer concentrations, and the correct storage of primer solutions. Multiplex PCR primer design guidelines are given in Table 10.

Table 10. General guidelines for multiplex PCR primer design
Length: 2130 nucleotides.
The probability that a primer has more than one specific binding site within a genome is significantly lower for longer primers. In addition, longer primers allow annealing at slightly higher temperatures where Taq DNA polymerase activity is higher. GC content: 4060% Tm: >= 60C

Safety Information

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs).

24-hour emergency information
Emergency medical information in English, French, and German can be obtained 24 hours a day from:
Poison Information Center Mainz, Germany
Tel: +49-6131-19240

Product Use Limitations

The QIAGEN Multiplex PCR Kit is developed, designed, and sold for research purposes only. It is not to be used for human diagnostic or drug purposes or to be administered to humans unless expressly cleared for that purpose by the Food and Drug Administration in the USA or the appropriate regulatory authorities in the country of use. All due care and attention should be exercised in the handling of many of the materials described in this text.
Simplified formula for estimating melting temperature (Tm): Tm = 2C x (number of [A+T]) + 4C x (number of [G+C]) Whenever possible, design primer pairs with similar Tm values. For optimal results, we recommend using primer pairs with a Tm of >= 68C. Above 68C, differences in Tm values of different primer pairs do not usually affect performance. Check functionality and specificity of all primer pairs in individual reactions before combining them in a multiplex PCR assay. Sequence: Avoid complementarity of 2 or 3 bases at the 3' ends of primer pairs to reduce primer-dimer formation.
Avoid mismatches between the 3' end of the primer and the target-template sequence.
Avoid runs of 3 or more G and/or C at the 3' end.
Avoid complementary sequences within a primer sequence and between the primer pair.
Commercially available computer software (e.g., OLIGO 6, Rychlik, 1999) or web-based tools such as Primer3, Steve
Rosen & Helen Skaletsky, 2000, (http://wwwgenome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) can be used for primer design.
Ensure primer sequence is unique for your template sequence. Check similarity to other known sequences using BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Compatible
PCR products:
Depending on your method of detection, PCR products should be chosen so that they can be easily distinguished, for example by size differences of the corresponding PCR products or by using primers labeled with different fluorescent dyes.

3. Handling and storage of primers

Primer quality is crucial for successful multiplex PCR. Problems encountered with multiplex PCR are frequently due to incorrect concentrations of primers being used. If you observe large differences in yield of different amplification products in a multiplex PCR, check that all primers were used at the correct concentration. For optimal results, we recommend only combining purified primers of comparable quality.

Table 11. General guidelines for handling and storage of PCR primers
Primer storage buffer: Lyophilized primers should be dissolved in a small volume of low salt buffer to make a concentrated stock solution. We recommend using TE (10 mM TrisCl, 1 mM EDTA, pH 8.0) for standard primers and primers labeled with most fluorescent dyes. However, primers labeled with fluorescent dyes such as Cy3, Cy3.5, Cy5, and Cy5.5, should be stored in TE, pH 7.0 since they tend to degrade at higher pH. Dissolving primers: Before opening tubes containing lyophilized primers, spin tubes briefly to collect all material at the bottom of the tube. To dissolve the primer, add the required volume of TE, mix, and leave for 20 minutes to let the primer completely dissolve. We do not recommend dissolving primers in water. Primers are less stable in water than TE and some primers may not dissolve easily in water. Storage: Primers should be stored in TE in small aliquots at 20C. Unmodified primers are stable under these conditions for at le ast one year and fluorescently labeled primers are usually stable under these conditions for at least 6 months. Repeated freeze/thaw cycles should be avoided since they may lead to primer degradation.
For easy and reproducible handling of the numerous primers used in multiplex PCR, we recommend the
preparation of a 10x primer mix containing all primers necessary for a particular multiplex PCR assay at
equimolar concentrations (see Table 1). Table 12. Procedure to determine primer concentration and quality
Concentration: Spectrophotometric conversion for primers: 1 A260 unit = 2030 g/ml To check primer concentration, the molar extinction coefficient (260) can be used: A260 = 260 x molar concentration of the primer If the 260 value is not given on the primer data sheet, it can be calculated from the primer sequence using the following formula: 260 = 0.89 x [(A x 15,480) + (C x 7340) + (G x 11,760) + (T x 8850)]
Example
Concentration of diluted primer: 1 M = 1 x 10-6 M Primer length: 24 nucleotides with 6 each of A, C, G, and T bases Calculation of expected A260: 0.89 x [(6 x 15,480) + (6 x 7340) + (6 x 11,760) + (6 x 8850)] x (1 x 10-6 )= 0.232 The measured A260 should be within +/ 30% of the theoretical value. If the measured A260 is very different to the theoretical value, we recommend recalculating the concentration of the primers, or having the primers resynthesized. Primer quality: The quality of 1830mers can be checked on a 15% denaturing polyacrylamide gel; a single band should be seen.
Fluorescently labeled primers
Fluorescent labels should be chosen so that they are compatible with your detection instrument. We recommend choosing fluorescent labels according to the manufacturer of your detection instruments instructions. See Table 11 for general guidelines on handling and storage of fluorescently labeled primers.

4. Number of PCR cycles

A cycling program usually consists of 2545 cycles, depending on the number of copies of the starting template and the sensitivity of the detection system used. Increasing the number of cycles does not necessarily lead to a higher yield of multiplex PCR product; instead it may increase nonspecific background and may lead to artifacts. Table 13 provides general guidelines for choosing the number of cycles. We recommend starting with the lowest number of cycles given for a particular template amount.

Table 13. General guidelines for choosing the number of PCR cycles



Analysis using fluorescence detection systems

Fewer PCR cycles are needed to generate sufficient product for analysis using fluorescence detection instruments, such as automatic sequencers based on gel or capillary electrophoresis. The optimal number of PCR cycles and the amount of PCR product that is used for analysis is dependent on the sensitivity of the detection system and should be determined individually. We recommend using 1012 fewer cycles than the lowest number given in Table 13 for a particular template amount. Too man y PCR cycles may lead to artifacts or additional products and should be avoided.

5. Separation of multiplex PCR products using agarose gel electrophoresis

The concentration of the agarose gel for separation of multiplex PCR products should be appropriate for the overall size of products generated and should be suitable for resolving the small size differences between PCR products. For optimal results, we recommend the use of 1x TAE buffer for preparation and running of the gel.

Table 14. General guidelines for choosing the percentage of agarose gel for separation of multiplex PCR products


* Efficient separation of PCR products differing in size by about 20 bp is usually possible using standard molecular-biologygrade agarose. For separation of fragments that differ in size by less than 20 bp, we recommend using high-resolution agarose, for example MetaPhor agarose (FMC Bioproducts). For more
information, visit www.cambrex.com.

6. Sensitive multiplex PCR assays

PCR can be performed to amplify and detect even a single copy of a nucleic acid sequence. However, amplification of such a low number of target sequences is often limited by the generation of nonspecific PCR products and primer-dimers. The combination of HotStarTaq DNA Polymerase and QIAGEN Multiplex PCR Buffer increases specificity both at the start of and during PCR, making the QIAGEN Multiplex PCR Kit well suited for highly sensitive PCR assays. Sensitivity can be further increased when using very low amounts of DNA (<= 20 copies), b y increasing the annealing time from 90 seconds to 3 minutes. However, some alleles may not be detectable when using low amounts of template DNA, due to the fact that not all target loci are present in the reaction. This is caused by stochastic fluctuation.

7. Guidelines for special multiplex PCR applications

Transgene detection
Genetically modified animals and plants can be generated to study the function of particular genes. Targeted mutations can be introduced that alter the function of the gene locus of interest, for example by inactivating or modifying its function. This allows research on the role of certain genes in complex biological processes. Modified genomic loci can be easily distinguished by multiplex PCR. To distinguish the wild-type from the mutant gene locus, pairs of primers should be designed that are specific either for the wild-type locus or for the mutant locus. In Figure 1, one primer is located on the wild-type locus. Two reverse primers have been designed, so that one primer is specific for the wild-type locus and the other specific for the mutant locus, resulting in PCR products of different sizes. Multiplex PCR is highly suited for the efficient detection of transgenes. In this case, two primer pairs can be used: one pair specific for the introduced transgene and the other pair specific for a wild-type DNA sequence. This second primer pair acts as a control for the amount and quality of the template DNA.

Detection of Transgenes


Figure 1. Transgenic mice were screened using the QIAGEN Multiplex PCR Kit and a set of 3 primers specific for the recombination activating gene 2 locus. The primers were designed to generate PCR products of different sizes so that wild-type mice (wt) could be easily distinguished from heterozygote mutant mice (ht), or homozygote mutant mice (hm).

Analysis of microsatellites

Eukaryotic genomes contain repeated DNA sequences that differ both in the length and sequence of the repeat. These repeats, known as satellite DNA, are often highly polymorphic, with many different alleles present within a population. Alleles may also differ in the number of copies of a particular repeat sequence. Variation in satellite DNA sequences is important and can be used to determine genetic differences between organisms or closely related individuals. Satellite loci used for PCR analysis are usually short repeat units known as microsatellites. The length of the repeat unit is typically 26 nucleotides, and is referred to as a short tandem repeat (STR). In some cases, slightly longer repeats are used for analysis. These are referred to as variant number of tandem repeats (VNTRs) and can vary in both repeat length and number of repeats.

Amplification of microsatellite DNA may result in a low percentage of PCR products that are shorter than expected. Typically, the difference in size of these "stutter products" is exactly one repeat unit and these products are an intrinsic feature of the amplification of some satellite DNA sequences.

Taq DNA polymerases, including HotStarTaq in the QIAGEN Multiplex PCR Master Mix, add an extra A residue to the 3'-end of PCR products. When using the microsatellite cycling protocol, all PCR products should have this additional A residue at the 3' end. However, if larger amounts of PCR product are generated using an increased number of cycles, it may be possible that not all PCR products will have the extra A residue and some n-1 product may be detectable. This may interfere with data analysis, depending on the resolution of the detection system. Decreasing the number of cycles can help to minimize the number of n-1 products.

Multiplex PCR of microsatellite loci is frequently performed using fluorescently labeled primers. Ensure that the fluorescent labels are compatible with the detection system used. Different fluorescent dyes may give differing signal intensities on a particular detection instrument, although comparable amounts of PCR product are generated. We recommend combining fluorescent dyes for multiplex PCR according to the instructions of the detection instrument's manufacturer.

Analysis of microsatellite DNA is usually performed using nanogram amounts of template DNA. However, in some cases only smaller amounts of DNA may be available. The QIAGEN Multiplex PCR Kit is highly suited for performing microsatellite analysis with low amounts (<= 0.1 ng) of genomic DNA. Increasing the annealing time from 90 seconds to 3 minutes can further increase the sensitivity of the reaction, and gives very reliable genotyping results. With such low amounts of template, some alleles may not be detectable due to the fact that not all target loci are present in the reaction. This is caused by stochastic fluctuation.

SNP analysis

Single nucleotide polymorphisms (SNPs) are single nucleotide changes occurring at specific points within the genome. Analysis of SNPs is important, because they are a genetic marker that can be used to associate genetic changes with disease. Study of SNPs can also be used to analyze complex phenotypes or to establish whether a genetic relationship exists between certain individuals. To provide sufficient material for analysis, the regions carrying the relevant SNPs can be amplified from genomic DNA by multiplex PCR, using the QIAGEN Multiplex PCR Kit. After amplification, primers, unincorporated nucleotides, enzyme, salts, and mineral oil may need to be removed before the specific PCR products can be used in subsequent experiments. The QIAquick System offers a quick
and easy method for purifying PCR products. Using the MinElute System, PCR products can be purified in higher concentrations due to the low elution volumes needed in this system. For more information about QIAquick and MinElute products, call your local QIAGEN Technical Service Department or distributor (see inside front cover for contact information).

Detection of genetically modified organisms or microorganisms

Multiple organisms can be detected simultaneously using multiplex PCR. In comparison to established methods, the QIAGEN Multiplex PCR Kit provides higher sensitivity of amplification due to the specially optimized QIAGEN Multiplex PCR buffer in combination with HotStarTaq polymerase. This allows amplification of multiple products with differing copy numbers in the multiplex PCR reaction. See Appendix for more information on amplification of sequences from low amounts of DNA and also for information on sensitive multiplex PCR assays.

Exon-specific PCR

Multiplex PCR is frequently used for amplification of multiple regions of a gene, for example to detect regions carrying SNPs or other mutations. Since the outside primers in the multiplex PCR reaction may still be quite close to each other, additional larger PCR products may be amplified from these outside primers (Figure 2).

Generation of Additional PCR Products in Exon-Specific PCR



Figure 2. Two exons from the human HFE gene were amplified from human genomic DNA either separately, (Exon 4); (Exon 2), or together (Exon 2 and 4). The PCR products generated from Exon 4 and Exon 2 were 395 bp and 242 bp respectively. However, in the multiplex PCR an additional product of 2219 bp is observed, generated by the 2 outside primers.

8. Optimization of reaction conditions for special multiplex PCR applications

The QIAGEN Multiplex PCR Kit protocols have been developed to give satisfactory results in most cases. In some special cases, modifications to the conditions given in the protocol may improve performance.

Large number of PCR products: For multiplex PCR reactions with more than 10 PCR products, a decrease of primer concentration to 0.1 M may lead to a more uniform product yield. The annealing time can also be increased from 90 seconds to 3 minutes. To establish a multiplex system with a large number of PCR products, it is strongly recommended to check the primer concentration given by the primer supplier. The use of high-quality primers, for example HPLC purified, is recommended, although standard quality may also be sufficient. We strongly recommend only combining primers of comparable quality. Highly sensitive applications or
low template amount:
Increasing the annealing time from 90 sec r> Product Warranty and Satisfaction Guarantee

QIAGEN guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, QIAGEN will replace it free of charge or refund the purchase price. We reserve the right to change, alter, or modify any product to enhance its performance and design. If a QIAGEN product does not meet your expectations, simply call your local Technical Service Department or distributor. We will credit your account or exchange the product as you wish.

Introduction

The QIAGEN Multiplex PCR Kit is specifically developed for multiplex PCR. It eliminates the need for optimization, making the development of multiplex PCR assays both simple and fast. The kit contains a master mix specially designed for multiplex PCR applications. The master mix contains pre-optimized concentrations of HotStarTaq DNA Polymerase and MgCl2, plus dNTPs, and a PCR buffer newly developed for multiplex PCR reactions. Use of a master-mix format reduces time and handling for reaction setup and increases reproducibility by eliminating many possible sources of pipetting errors. The kit is well suited for multiplex PCR applications such as typing of genetically modified animals and plants, microsatellite analysis, determination of bacteria and viruses, or amplification of regions carrying SNPs.

HotStarTaq DNA Polymerase

The QIAGEN Multiplex PCR Master Mix contains HotStarTaq DNA Polymerase, a modified form of QIAGEN Taq DNA Polymerase. HotStarTaq DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primer-dimers during reaction setup and the first denaturation step, leading to exceptionally high PCR specificity. HotStarTaq DNA Polymerase is activated by a 15-minute, 95C incubation step, which is easily incorporated into existing thermal cycling programs. The hot start enables reactions to be set up at room temperature, making setup rapid and convenient. (Refer: HotStarTaq PCR Handbook)

QIAGEN Multiplex PCR Buffer

The unique QIAGEN Multiplex PCR Buffer facilitates the amplification of multiple PCR products. In contrast to conventional PCR reagents, the QIAGEN Multiplex PCR Buffer contains a specially developed balanced combination of salts and additives to ensure comparable efficiencies for annealing and extension of all primers in the reaction. Lengthy optimization procedures, such as adjusting the amounts of Taq DNA polymerase, Mg2+, additional reagents, and primers is virtually eliminated.

Q-Solution

The QIAGEN Multiplex PCR Kit is provided with Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting beh avior of DNA. This unique reagent often enables or improves a suboptimal PCR caused by templates that have a high degree of secondary structure or that are GC-rich. Unlike other commonly used PCR additives, such as DMSO, Q-Solution is used at just one working concentration, which has been specially optimized for the requirements of multiplex PCR. It is nontoxic, and PCR purity is guaranteed. For further information, please read the protocol using Q-Solution.

Product Description

2x QIAGEN Multiplex PCR Master Mix contains:
HotStarTaq DNA Polymerase: HotStarTaq DNA Polymerase is a modified form of a recombinant 94 kDa DNA polymerase, originally isolated from Thermus aquaticus, cloned in E. coli. (Deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) QIAGEN Multiplex PCR Buffer: Contains 6 mM MgCl2, pH 8.7 (20C) dNTP Mix: Contains dATP, dCTP, dGTP, dTTP;
ultra pure quality Q-Solution: 5x concentrated Distilled water: Ultrapure quality, PCR-grade

Quality Control

QIAGEN Multiplex PCR Master Mix
Multiplex PCR performance assay: PCR performance, reproducibility, and sensitivity are t ested in parallel multiplex PCR reactions. HotStarTaq DNA Polymerase: (included in QIAGEN Multiplex PCR Master Mix) Amplification efficiency and reproducibility in PCR are tested. Functional absence of exonucleases and endonucleases is tested. Buffers and reagents QIAGEN Multiplex PCR Buffer: Density, pH, and content of cationic components are tested. Q-Solution, 5x: Conductivity, density, pH, and performance in PCR are tested. Distilled water: Conductivity, pH, and RNase activities are tested.

Solutions and Reagents to Be Supplied by User

Primers
The QIAGEN Multiplex PCR Kit can be used with standard-quality primers that can be purchased from established oligonucleotide manufacturers. Primers should be purchased desalted or purified, for example using reverse-phase purification, HPLC purification, or related purification technologies and dissolved in TE (10 mM TrisCl, 1 mM EDTA, pH 8.0).
Important note:
It is strongly recommended to test the functionality and specificity of all primer pairs in single reactions before combining them in a multiplex PCR assay. See Appendix for guidelines on multiplex PCR primer design.

For easy and reproducible handling of the numerous primers used in multiplex PCR, we recommend the preparation of a primer mix containing all primers at equimolar concentrations. The primer mix should be prepared in TE, as described in Table 1, and stored in small aliquots at 20C to avoid repeated freezing and thawing. Multiple freeze/thaw cycles of the primer mix may lead to decreased assay performance.

Table 1. Preparation of 10x primer mix (containing 2 M each primer)*

* Allows preparation of a 10x primer mix containing up to 12 primer pairs (50 M stock) or containing up to 25 primer pairs (100 M stock).

Standard Multiplex PCR Protocol

Important notes before starting
  • Always start with the cycling conditions specified in this protocol.
  • If using an already established multiplex PCR system, use the previously established annealing temperature in combination with the cycling conditions specified in this protocol.
  • Annealing time must be 90 s.
  • Use equal concentrations (0.2 M) of all primers.
  • PCR must start with an activation step of 15 min at 95C to activate HotStarTaq DNA Polymerase (see step 7 of this protocol).
  • Optional: If a thermal cycler with a temperature gradient function can be used, determine the optimal annealing temperature by performing a gradient PCR.

Protocol

1. Thaw 2x QIAGEN Multiplex PCR Master Mix (if stored at 20C), template DNA, distilled water, and primer mix. Mix the solutions completely before use.
It is important to mix the solutions completely before use to avoid localized concentrations of salts. Preparing a mixture of all primers avoids pipetting of individual primers for each experiment, reducing pipetting time and increasing reproducibility of results (for preparation of primer mix see Table 1). 2. Prepare a reaction mix according to Table 2
The reaction mix typically contains all the components required for multiplex PCR except the template DNA. Prepare a volume of reaction mix 10% greater than that required for the total number of reactions to be performed. For reaction volumes less than 50 l, the 1:1 ratio of QIAGEN Multiplex PCR Master Mix to primer mix and template should be maintained as shown in Table 2.
Note: We strongly recommend starting with an initial Mg2+ concentration of 3 mM as provided by the 2x QIAGEN Multiplex PCR Master Mix. Table 2. Multiplex PCR components (reaction mix and template DNA)


* Provides a final concentration of 3 mM MgCl2.
A final primer concentration of 0.2 M is optimal for most primertemplate systems. However, in some cases using other primer concentrations (0.10.3 M) may further improve amplification performance.
For volumes less than 50 l, the 1:1 ratio of QIAGEN Multiplex PCR Master Mix to primer mix and template should be maintained. 3. Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tubes or plates.
Mix gently, for example by pipetting the reaction mix up and down a few times. Due to the hot start, it is not necessary to keep samples on ice during reaction setup.
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