PowerPlex® Fusion System

3.

After completion of the thermal cycling protocol, store amplified samples at –20°C in a light-protected box.

Note:

Long-term storage of amplified samples at 4°C or higher may produce artifacts.

4.B. Direct Amplification of DNA from Storage Card Punches

Materials to Be Supplied by the User

• GeneAmp ® PCR System 9700 thermal cycler (Applied Biosystems)

• microcentrifuge

• MicroAmp ® optical 96-well reaction plate or 0.2ml MicroAmp ® reaction tubes (Applied Biosystems)

• aerosol-resistant pipette tips

• PunchSolution™ Kit (Cat.# DC9271) for nonFTA card punches

• 1.2mm Harris Micro-Punch or equivalent manual punch and cutting mat or automated punch system

This section contains a protocol for direct amplification of DNA from storage card punches using the PowerPlex

®

9700 thermal cycler.

Fusion System and GeneAmp

®

PCR System

We recommend amplifying one or two 1.2mm punches of a storage card containing a buccal sample or one 1.2mm punch of a storage card containing whole blood in a 25µl reaction volume using the protocols detailed below. The

PowerPlex ® Fusion System is optimized for the GeneAmp ® PCR System 9700 thermal cycler.

Note:

You will need to optimize and validate the number of storage card punches per reaction in your laboratory. See the PCR optimization recommendations at the end of the section.

FTA

®

-based sample types include:

• Buccal cells collected on FTA ® cards with Whatman EasiCollect™ or Fitzco

Sampact™ devices

• Buccal cells collected with sterile swabs transferred to FTA ® or Indicating

FTA ® cards

• Liquid blood (from collection or storage Vacutainer ® tubes or finger sticks) spotted onto FTA ® cards

NonFTA sample types include:

• Buccal samples on Bode Buccal DNA Collector™ devices

• Blood and buccal samples on nonFTA card punches (e.g., S&S 903)

Pretreat nonFTA sample types with the PunchSolution™ Kit (Cat.# DC9271) to lyse nonFTA samples before adding the PCR amplification mix. For more information, see the PunchSolution™ Kit Technical Manual #TMD038. Failure to pretreat these samples may result in incomplete profiles.

Use a manual punch tool with a 1.2mm tip to manually create sample disks from a storage card. Place tip near the center of the sample spot, and with a twisting or pressing action, cut a 1.2mm sample disk. Use the plunger to eject the disk into the appropriate well of a reaction plate.

Promega Corporation ·

2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com

Printed in USA.

Revised 10/12

Part# TMD039

Page 9

4.B Direct Amplification of DNA from Storage Card Punches (continued)

Automated punchers also can be used to create sample disks. Refer to the user’s guide for your instrument for assistance with generating 1.2mm disks, technical advice and troubleshooting information.

Note:

Static may be problematic when adding a punch to a well. For FTA ® card punches, adding PCR amplification mix to the well before adding the punch may help alleviate static problems. For nonFTA card punches, adding

PunchSolution™ Reagent to the well before adding the punch during pretreatment may help alleviate static problems.

Amplification Setup

1.

Thaw the PowerPlex ® Fusion 5X Master Mix, PowerPlex ® Fusion 5X

Primer Pair Mix and Water, Amplification Grade, completely.

Note:

Centrifuge tubes briefly to bring contents to the bottom, then vortex reagents for 15 seconds before each use. Do not centrifuge the 5X Primer

Pair Mix or 5X Master Mix after vortexing, as this may cause the reagents to be concentrated at the bottom of the tube.

2.

Determine the number of reactions to be set up. This should include positive and negative control reactions. Add 1 or 2 reactions to this number to compensate for pipetting error. While this approach does consume a small amount of each reagent, it ensures that you will have enough PCR amplification mix for all samples. It also ensures that each reaction contains the same PCR amplification mix.

3.

Use a clean MicroAmp ® plate for reaction assembly, and label appropriately. Alternatively, determine the number of clean, 0.2ml

reaction tubes required, and label appropriately.

4.

Add the final volume of each reagent listed in Table 2 to a sterile tube.

Table 2. PCR Amplification Mix for Direct Amplification of DNA from Storage Card

Punches.

PCR Amplification Mix

Component

PowerPlex ®

1

Water, Amplification Grade

Fusion 5X Master Mix

PowerPlex ® Fusion

5X Primer Pair Mix

Volume

Per Reaction

15µl

5.0µl

5.0µl

×

×

×

×

Number of

Reactions

=

=

=

=

Final

Volume total reaction volume

25µl

1 Add Water, Amplification Grade, to the tube first, then add PowerPlex

Mix and PowerPlex ® Fusion 5X Primer Pair Mix. For FTA

DNA will be aded at Step 6.

®

® Fusion 5X Master card punches, the template

Promega Corporation ·

2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com

Part# TMD039

Page 10

Printed in USA.

Revised 10/12

5.

Vortex the PCR amplification mix for 5–10 seconds, then pipet 25µl of PCR amplification mix into each reaction well.

!

Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus-to-locus imbalance.

6.

For FTA ® storage cards, add one or two 1.2mm punches from a card containing a buccal sample or one 1.2mm punch from a card containing whole blood to the appropriate wells of the reaction plate. For nonFTA card punches, add the PCR amplification mix to the PunchSolution™

Reagent-treated punches.

Note:

It also is acceptable to add the FTA ® card punch first, then add the

PCR amplification mix.

7.

For the positive amplification control, add 1μl of 2800M Control DNA

(10ng) to a reaction well containing 25μl of PCR amplification mix.

Notes:

1.

Do not include blank storage card punches in the positive control reactions.

2.

Optimization of the amount of control DNA may be required, depending on cycling conditions and laboratory preferences.

8.

Reserve a well containing PCR amplification mix as a negative amplification control.

Note:

An additional negative control with a blank punch may be performed to detect contamination from the storage card or punch device.

9.

Seal the plate, and briefly centrifuge the plate to bring storage card punches to the bottom of the wells.

Thermal Cycling

Amplification and detection instrumentation may vary. You will need to optimize protocols including the number of storage card punches, cycle number

(25–28 cycles), injection time and loading volume for your laboratory instrumentation. Testing at Promega shows that 27 cycles works well for a variety of sample types. Buccal samples may require more amplification cycles than blood samples. Cycle number should be optimized in each laboratory for each sample type that is amplified.

1.

Place the MicroAmp ® plate in the thermal cycler.

Promega Corporation ·

2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com

Printed in USA.

Revised 10/12

Part# TMD039

Page 11

4.B Direct Amplification of DNA from Storage Card Punches (continued)

2.

Select and run the recommended protocol. Be sure that Max mode is selected as the ramp speed. The preferred protocol for use with the

GeneAmp ® PCR System 9700 thermal cycler is provided below. The estimated total cycle time is 1.5 hours.

Thermal Cycling Protocol 1

96°C for 1 minute, then:

94°C for 10 seconds

59°C for 1 minute

72°C for 30 seconds for 27 cycles, then:

60°C for 20 minutes

4°C soak

1 When using the GeneAmp ® PCR System 9700 thermal cycler, the program must be run with Max mode as the ramp speed. (This requires a silver or gold-plated silver sample block.) The ramp speed is set after the thermal cycling run is started. The Select Method Options screen appears.

Select “Max” for the ramp speed, and enter the reaction volume.

Note:

The final extension for direct amplification was extended to

20 minutes compared to 10 minutes for the extracted DNA protocol to allow sufficient time for adenylation of large amounts of amplicon.

3.

After completion of the thermal cycling protocol, store amplified samples at –20°C in a light-protected box.

Note:

Long-term storage of amplified samples at 4°C or higher may produce artifacts.

PCR Optimization

Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method, sample types, number of punches and instrumentation.

1.

Choose several samples that represent typical sample types you encounter in the laboratory. Prepare them as you would using your normal workflow.

2.

Depending on your preferred protocol, place one or two 1.2mm storage card punches containing a buccal sample or one 1.2mm punch of a storage card containing whole blood in each well of a reaction plate. Be sure to pretreat nonFTA samples with the PunchSolution™ Kit (Cat.# DC9271).

3.

Prepare four identical reaction plates with punches from the same samples.

4.

Amplify samples using the thermal cycling protocol provided above, but subject each plate to a different cycle number (25–28 cycles).

5.

Following amplification, use your laboratory’s validated separation and detection protocols to determine the optimal cycle number for the sample type and number of storage card punches.

Promega Corporation ·

2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com

Part# TMD039

Page 12

Printed in USA.

Revised 10/12