2. Pyrosequencing Assay Design

Transcription

1 2. Pyrosequencing Assay Design 2.1 Guidelines for PCR set-up and primer design PCR primer design Design of PCR primers follows standard rules, i.e. calculated Tm of C, primer length of about bases, check of dimer and loop formation, relatively equal G/C A/T distribution within the primers etc. One primer must be biotin-labeled (for immobilization to streptavidin-coated magnetic- or Sepharose beads), the other unlabeled, see Figure 2-1. We strongly recommend purification of the biotinylated primer by HPLC, or equivalent procedure, in order to minimize the amount of free biotin and maximize the proportion of biotinylated primer. After NaOH denaturation, and annealing of the sequencing primer the immobilized strand can be sequenced. DNA denaturation and PCR primer annealing PCR Strand separation Sequencing primer annealing Figure 2-1. Schematic view of PCR using one biotinylated () primer, sample preparation and sequencing primer ( ) annealing Length of PCR products For SNPs, where the PCR amplicon size can be directed by primer design, as short PCR products as possible are recommended, preferably less than 200 bp. Nevertheless, up to 500 bp long fragments have been tested with good results, and even longer work for some assays. Smaller fragments compared to longer have several advantages. The amplification is easier, the SNP resolution is improved, and for magnetic streptavidin-coated beads, the consumption of beads is reduced (see Section Sample preparation using Dynabeads M-280 Streptavidin) PCR reaction The standard PCR protocol used at Pyrosequencing A is shown to the right for a 50 µl PCR reaction. 1X PCR buffer 10 pmol downstream primer 10 pmol upstream primer 1 U AmpliTaq Gold (Applied iosystems) mm of each dntp mm MgCl 2 * 10 ng template *The MgCl 2 -concentration has to be titrated 1

2 2.1.4 PCR cycling conditions The PCR cycling program has to be optimized. At an optimal annealing temperature, up to 50 cycles can be run. A standard cycling program run at Pyrosequencing A with PCR reaction components as described above is shown to the right. 95 C 5 min 50 x (95 C 15 s, T a 30 s, 72 C s) 72 C 5 min 4 C Sequencing primer design Design of sequencing primers for Pyrosequencing TM follows the same criteria as for the PCR primers, except that the Tm of this primer may, if necessary, be lowered. This usually means that the sequencing primer can be made shorter than the PCR primers, typically 15 bp. ased on our current experience, primers with a Tm around 50 ºC work well in most cases. The lowest possible Tm is still not defined, but primers with Tm around 40 ºC are frequently used with good results. The sequencing primer should be unlabeled. As the sequencing reaction is run at 28 ºC, it is crucial to check the primer for self-annealing, especially at the 3 -end. We have seen problems with as few as 4 matching terminal 3 bases and also with as few as 1 terminal 3 -match if the dimer is stabilized at other positions. A problem that might arise is that background peaks will be generated by extension of the recessed 3 -end of the self-annealed sequencing primer (Figure 2-2) GGCGGATCGATGACGAT TAGCAGTAGCTAGGCGG---5 Figure 2-2. Peaks generated by extension of the recessed 3 -end of a self-annealed sequencing primer. Note: The sequencing primer should always be complementary to the biotinylated strand. Sequencing primer for SNP genotyping and allele quantification In case of SNP sequencing, the position of the sequencing primer is flexible within 5-15 bases from the polymorphic position. Also consider that the SNP can be sequenced on either strand. The interpretation of the SNP might be difficult if one or both of the polymorphic bases will form a homopolymer with adjacent bases. Therefore, try to design your sequencing primer to avoid such a situation by placing the 3 -end to overlap the polymeric stretch, see Figure 2-3. However, if this is impossible, homopolymers of up to 5 C, G, or T or 3 A are acceptable. 2

3 As the sequencing reaction is run at 28 ºC, it is crucial to check the primer for self-annealing, especially at the 3 -end. We have seen problems with as few as 4 matching terminal 3 bases and also with as few as 1 terminal 3 -match if the dimer is stabilized at other positions. A problem that might arise is that background peaks will be generated by extension of the recessed 3 -end of the self-annealed sequencing primer, see Figure CTAAGTGGACGTGAAGCA 5 -AGTGGACGTGAAGCAAAA 3 -CCGAGTGATTCACCTGCACTTCGTTTT T/CACGAATGC-5 Figure 2-3. Preferred positioning of the sequencing primer for a polymorphic position situated within a homopolymeric stretch. For optimal sequencing primer design, please use our on-line SNP primer design software at: Sequencing primer for SQA analysis For SQA analysis, the positioning of the primer should be as close as possible to the sequence to be read. This will allow for maximum read lengths. Nonetheless, it is recommended to start sequencing with 2-3 known bases, preferably single bases. These bases, as well as known sequence motifs anywhere along the sequence, can be utilized by the algorithm to call the correct sequence. 3

4 2.2 PCR optimization - considerations for use of Pyrosequencing technology 1. Select PCR primers, which form, if possible, a fragment 200 bp. The PCR primers should typically be about bp in length, of approximately the same GC-content as the fragment as a whole, and with approximately the same melting temperatures. The primers should preferably be more GC-rich in the 5 -end and less in the 3 -end for good specificity. They should not form heavy hairpin loops or dimers with themselves or the other primer. Check the biotinylated PCR primer extra carefully for hairpin loops and duplexes, as excess biotinylated primer might cause background in the Pyrosequencing assay. 2. If Oligo 6.0 ( is used for primer design, you can let Oligo find a primer set automatically. The program will also present calculated optimal and highest annealing temperatures for the selected primer set, and a good starting point is to choose an annealing temperature between these values. 3. iotinylated PCR primer concentrations should be kept low to avoid interference with the Pyrosequencing assay. Keep the primer concentrations at 0.2 µm (i.e. 10 pmol in a 50 µl PCR reaction). 4. iotinylated PCR primers are particularly sensitive to storage. Keep stock primer, and aliquoted, diluted primers (10 µm), in the freezer, not in the fridge! 5. The effective, free magnesium concentration will depend on nucleotide and DNA concentrations (because magnesium binds to nucleotides and DNA). Therefore, keep DNA and nucleotide concentrations constant during PCR optimization. As a standard, we use 10 ng DNA in a 50 µl PCR reaction and 125 µm of each nucleotide. 6. In general, increased magnesium concentrations will lead to higher incorporation rate and efficiency for AmpliTaq Gold, but may also make the enzyme slightly more sloppy, and increase the risk for incorporation errors. Furthermore, it will stabilize dimers, increasing the risk for mis-priming in the template, as well as secondary structures in the DNA template, which may decrease the amplification efficiency for GC-rich templates in particular. Lowered magnesium concentrations will in general make the amplification reaction more stringent, but also less efficient, leading to lower yields. 7. AmpliTaq Gold is gradually activated during the amplification reaction and therefore requires more cycles than a protocol with ordinary Taq. For best yield, and consumption of all the biotinylated (PCR) primer which is important to avoid background arising from the biotinylated primer, run PCR cycles. 8. The parameters that may need optimization are the annealing temperature and magnesium concentration. The annealing temperature typically falls in the range 54 C 64 C. Select two DNA samples that can be used for all PCR optimizations. With AmpliTaq Gold, a good starting point is to try three different temperatures (e.g. 54 C, 57 C, and 60 C) and three different magnesium concentrations (1.5 mm, 2.0 mm, and 2.5 mm) while keeping all other parameters constant. GC-rich templates often need a higher annealing temperature and lower magnesium concentration (since high salt concentrations will stabilize secondary structures) to amplify well. Very often, medium GC-rich templates will work at 57 C and 2.0 mm magnesium, so these might be conditions that you want to try before starting optimization. 9. A standard PCR program for fragments up to about 300 bp: 95 C 5min, 45 cycles (95 C 15s, Ta 30s, 72 C 15s), 72 C 5min, 4 C The program takes about 1 hour and 45 min to run. For longer fragments than 300 bp, the extension time at 72 C may need to be extended. 10. Check on 1.5% agarose that you have a clear, strong product band without excess primers, primer-dimers or other non-specific products. With 10 pmol of sequencing primer and µl of the PCR product, you could then expect strong, pure signals (single peak heights of ~15-25 units) when sequencing the products using a PSQ system. 4

5 Table 2-1. Example of PCR reaction mix (2.0 mm MgCl 2 ) for one and ten 50 µl reactions, respectively. The presented volumes are in microliters. Take 45 µl of reaction mix and 5 µl 2 ng/µl DNA per tube/well. PCR mix component 1 tube 10 tubes H 2 O x PCR buffer II (Applied iosystems) 5 50 MgCl 2 (25mM) 4 40 dntp (2.5 mm) Forward primer (10 µm) 1 10 Reverse primer (10 µm) 1 10 AmpliTaq Gold (Applied iosystems) Total: GLOAL TECHNICAL SUPPORT: PHONE FAX techsupport.se@pyrosequencing.com US TECHNICAL SUPPORT: PHONE FAX techsupport.us@pyrosequencing.com PYROSEQUENCING A VALLONGATAN 1, SE UPPSALA, SWEDEN PHONE , FAX