Frequently Asked Questions

1. How do I use your kit to detect Single Nucleotide Polymorphisms (SNPs)?

Use the AcycloPrime-FP kit to detect known SNPs from genomic DNA after you have amplified the region of DNA that has the SNP of interest.

2. Is it possible to use this kit for discovering SNPs?

SNPs are discovered by de novo Sanger sequencing. Since one needs to have knowledge of sequence information in the area of the SNP to be able to design both the PCR primers and the SNP primer, this method can not be used to discover SNPs.

3. What are the steps in performing the AcycloPrime assay?

• Isolation & amplification of genomic DNA (PCR or other)
• Degradation of primers and free nucleotides using the PCR Clean-up Reagents provided in the AcycloPrime kit
• 3'-End labeling of the SNP primer with dye-AcycloTerminator(s) using AcycloPol
• Detection of incorporated dye-AcycloTerminators by fluorescence polarization (FP) using the Victor2 or another instrument.

4. Has the method been published?

• Chen, X, Levine, L, and Kwok, P-Y., "Fluorescence polarization in homogeneous nucleic acid analysis", Genome Res. (1999) 9, 492-498.
• Hsu, T.M., Chen, X., Duan, S., Miller, R.D., and Kwok, P-Y., "Universal SNP genotyping assay fluorescence polarization detection", BioTechniques (2001) 31, 560-570.
• US Patent 6,180,408 issued to Wash. Univ.

5. What licenses are conveyed with the purchase of the AcycloPrime-FP kit?

• A license for RESEARCH use, for the specified number of assays, under US Patent 6,180,408 (Wash. University, St. Louis) and US Patents 5,888,819 and 6,004,744 (Orchid BioSciences, Inc.)
• At this time, no fee-for-service or diagnostic testing is permitted.

6. What are the catalog numbers for AcycloPrimeT-FP SNP Detection Kit?

7. What are the reagents provided in the AcycloPrime-FP SNP kit?

• Newly upgraded entire kit offering, Feb. 15, 2002
  • PCR Clean-up Reagent, 10X (new)
  • PCR Clean-up Dilution Buffer (new)
  • 10X Reaction Buffer
  • AcycloTerminator Mix(es)
  • AcycloPol thermostable polymerase
• Please note: the kit name is the name of the labeled terminators within the kit. The first base in the name has the R110 dye and the second base the TAMRA dye.
• Reagents for (PCR) amplification are not provided.

8. Can the PCR Clean-Up Reagent, 10X, and PCR Clean-Up Dilution Buffer be purchased separately?

NO

9. What are the new "combo" kits: ACP113B and ACP113C?

• Using a single catalog number researchers can now obtain a mixture of all six 2-base AcycloTerminator combinations
• The ACP113B provides 10,000 total reactions by including:
  • 3 x 1,000 G/C; 3 x 1,000 C/T; 1,000 G/C; 1,000 G/T; 1,000 C/A; and 1,000 A/T
  • 10,000 reaction equivalent bottles of the rest of the kit components: PCR Clean-up Reagent, 10X/Diluent, AcycloPol, 10X Reaction Buffer
• The ACP113C provides 100,000 total reactions by including:
  • 3 x 10,000 G/C; 3 x 10,000 C/T; 10,000 G/C; 10,000 G/T; 10,000 C/A; and 10,000 A/T
  • 100,000 reaction equivalent bottles of the rest of the kit components: PCR Clean-up Reagent, 10X/Diluent, AcycloPol, 10X Reaction Buffer

10. Can the customer specify different terminator combinations?

NOT AT THIS TIME.

11. How is the assay formatted?

• Amplify Genomic DNA containing SNP site of interest
  - Use 5 ?L reaction volume, 5 ng DNA
• Degrade Primers and dNTPs, Inactivate Enzymes
  - Add 2 ?L of PCR Clean-Up Reagent to the PCR wells.
  - Incubate at 37°C for 1 hour
  - Heat to 80°C for 15 min.
• Primer Extension

- Thermal Cycling -

(1) Denature at 95°C for 2 minutes.
(2) Perform 10-40 cycles of 95°C for 15 sec. and 55°C for 30 sec.
(3) At end of cycles ramp down to 15°C for 2 minutes then to 4°C.

12. What are the main advantages of this assay?

• The homogeneous assay requires no centrifugation, separation, immobilization or wash steps. Easy to automate.
• Unlabeled primers - inexpensive and rapid setup
• Only 5 ng of genomic DNA is usually sufficient.
• Validated to-date to work on over 10,000 different SNPs
• Affordable

13. Can the AcycloPrime-FP be used on other instruments?

• Yes. The MD/LJL Analyst and the Tecan Ultra have been validated in addition to the PerkinElmer VICTOR²
• Please ensure you have the correct filters and dichroic mirrors for the excitation/emission wavelengths of R110 and TAMRA.

4. Why are you offering only 2 dye-AcycloTerminator mixes?

• We feel at this time the assay is more ROBUST (both from chemistry and instrument) with only 2 colors vs. a 4-color format
• We understand there is more up-front thought required to organize which SNPs per plate or which AcycloPrime mix per well
• We continue to evaluate the feasibility of a 4-color format

15. Do you have any application notes for this method?

Yes. Request Application note # SNP01, rev. A, 032001

16. How do I store the kit?

• The kit should be stored at -20ºC for maximum shelf life
• The kit is shipped on dry ice
• Some component may be stored at 4ºC for convenience
• Ensure AcycloTerminators are stored in the DARK. Limited light exposure is tolerated for routine assay procedures
• AcycloTerminator Mixes are now shipped in brown bottles

17. What microplate is recommended?

• Black PCR plates, such as MJ Research cat. No. MSP-3862 (384 wells) or HSP-9666 (96 wells)
• 96 well plates may need 1 µL of additional water per well because of evaporation

18. What is an AcycloTerminator?

• AcycloTerminators are novel structures which have been
  thoroughly validated and shown to behave similarly to the
  commonly-used ddNTP terminators
• AcycloPol preferentially incorporates AcycloTerminators
  over dideoxynucleotides and also prefers labeled
  AcycloTerminators to unlabeled ones

19. What is AcycloPol?

• AcycloPol is a novel, mutant thermostable DNA polymerase from the Archeon family of bacteria
• It is not related to ThermoSequenase (APB) or AmpliTaq FS (ABI) which are Taq mutants and have Phe:Tyr substitutions
• Gardner, A.F. and Jack, W.E. "Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases", Nucleic Acids Res. 30, 605-613.

20. Can I dilute the reaction to get more reactions than the kit was designed to provide?

• The kit is sold under licenses which define the number of SNP genotypes per kit, modification of the number goes against the license.
• As there are many variables which can effect the performance or success of this kit, we do not recommend or support changes to the reaction as defined.

21. What methods of amplification are recommended?

• At this time, PCR is the preferred method
• Reagents for the PCR process are not included in the AcycloPrime kit. Purchase a licensed kit from one of the many vendors currently providing these kits
• NOTE: this process is not only used to amplify the starting DNA but also results in reducing the genetic complexity of the sample thus aiding in specificity of the primer extension reaction.
• Recommendations for PCR:

• PCR primers must be designed and provided by the customer

22. What is the PCR Clean-up Reagent?

• The reagent is a mixture of two different enzymes:
  1) Exo = Exonuclease I which degrades single stranded DNA primers remaining after amplification
  2) SAP = Shrimp Alkaline Phosphatase which removes the phosphates from the dNTPs remaining after amplification.
• The reagent is sold under a license from USB Corp.
• This reagent has been optimized to degrade primers and dNTPs as recommended in AcycloPrime system.
• The reagent is substantially different than Exo-SAP-IT, USB Catalog Number 78201.
• Can alternatives be used? Maybe? The end user must optimize the any other method so as to ensure COMPLETE degradation of both primers and dNTPs.

23. Can AcycloPrime kits be obtained WITHOUT this reagent?

NO

24. What is the SNP primer??

• The SNP primer is a synthetic oligonucleotide that is complementary to the sequence adjacent to the location of the target SNP, thus the next base (e.g., the dye-AcycloTerminator) added to the primer will be complementary to the base at the SNP location.
• Using standard primer design techniques, SNP primers should be:
  • designed to minimize secondary structure
  • GC content about 50%
  • length ranging from 20-30 bases
  • a T_m in the range of 60°C-80°C.
• During assay validation for a specific allele, we recommend designing both the forward and reverse SNP primers and using the one that performs better. Performance is affected by the sequence context around the allele.

25. Which AcycloPrime kit should I use??

• Which bases are incorporated depends upon which orientation is chosen for the SNP primer. To illustrate, consider a hypothetical sequence for two heterozygous chromosomes containing a G/A SNP (bold) on the upper strand:
  • 5'-ACTTAGGCCGACCGCTAGCGTACAGAGGCCTTAGACATGCATGCAGT
  • 3'-TGAATCCGGCTGGCGATCGCATGTCTCCGGAATCTGTACGTACGTCA
  • 5'-ACTTAGGCCGACCGCTAGCGTACGGAGGCCTTAGACATGCATGCAGT
  • 3'-TGAATCCGGCTGGCGATCGCATGCCTCCGGAATCTGTACGTACGTCA
    
• If the SNP primer has the sequence underlined on the upper strand, the next base added will be either an A or a G, so use the G/A kit. If the SNP primer has the sequence underlined on the lower strand, the next base added will be either a C or a T, so use the C/T kit.

26. What support is currently offered by PerkinElmer for: PCR primer
design, SNP primer design, and data analysis?

• There are numerous free-ware programs available on the WEB developed by others for primer design.
• Data analysis:
  • Various Microsoft Excel spreadsheets have been made available for download free-of-charge from
  • MD(LJL) Analyst or Tecan Ultra version are available upon request.
  • Both 96- and 384-well plates can be used
  • Automatic data feed from the Victor to the Excel workbook.
  • Allele calling is semi-automatic after data inspection and setting of low/high cut-off values
  • Continue to check the WEB site for most recent updates and releases.

Allele Calling Software

27. What can you suggest for setting up the instrument(s) and or
measurement of FP?

• Proper filters must be installed in the instrument to measure the fluorescence of R110 (Ex 490 nm/Em 520 nm max) and TAMRA (Ex 550 nm/Em 570 nm max). Some instruments also require that the appropriate dichroic mirrors be installed.
• For the Victor2 V the recommended filters are:
  • R110             485_ex        535/25_em
  • TAMRA          544_ex         595/60_em
• Setting or adjusting of the "G" factor is highly recommended to facilitate troubleshooting and comparisons at different sites with different instruments. The G factor is a calibration that sets the mP reading of a solution of free terminator to 50. With the G-factor set to 1, read 20 µL of solution containing 1 µL of Terminator Mix and 2 µL of 10X Reaction Buffer. Insert the S and P intensities into the following equation and calculate the G-factor for each dye.

  G = 950S/1050P

• The FP value is calculated by following formula:

  mP = 1000 x [I_vv - I_vh]/[I_vv + I_vh]

  where I_vv is the emission intensity measured when the excitation and emission polarizers are parallel and I_vh is the emission intensity measured when the emission and excitation polarizers are oriented
  perpendicular to each other.

28. What are the currently known reasons for an assay FAILURE?

• Failed amplification reaction
• Over-amplified sample
  Reason: The AcycloPrime reaction uses an excess of SNP primer over dye-AcycloTerminators and thus once the "correct" terminators are used up the DNA polymerase will attempt to incorporate mis-matched bases.
• Inadequate sample cleanup after amplification
  Reason: It is critical that both the excess primers and dNTPs are removed from the reaction using Exo and SAP before performing the AcycloPrime step, remaining primers result in "false" sites being interrogated and dNTPs permit elongation longer than the single base addition desired.
• Over-cycling of the AcycloPrime reaction
  Reason: see explanation above for "Over Amplified sample". It is possible to perform stepwise cycling, e.g., 5-10 cycles then read FP, then repeat cycling if necessary.
• No polarization detected
  Reason: instrument problem, filter problem, one or more of above? Check to see if allele-specific failure, e.g., some wells positive and
  others negative or this is a total assay failure.