Additional notes on experimental protocols

The polony haplotyping protocol is based on previous protocols on polony experiments developed in our lab. Please read through the polony amplification protocol and single base extension protocol on our web site. I will only focus on the difference in the following discussions.

1. Chromosome isolation:
   

• RNase is not necessary for the chromosome isolation buffer. It doens't hurt either though.
• Often the chromosome isolation need to be passed through 22 gauge needle 8 times to achieve better cell lysis.
• YOYO-1 is superior to propidium iodide for chromosome staining. The staining can be done by adding 1ul of 1uM YOYO-1 to 50ul chromosome isolation and incubate at room temperature for 10 minutes.
• When chromosomes is too diluted, they can be concentrated by spinning at 5,000g  for 5 minutes in a 1.8ml Eppendorf tube, and removing part of supernatant.

2. Polony amplification:
• For the last wash in dH2O before amplification, adding ~10ul of Tween 20 (to ~40ml of dH2O) really helps avoiding air bubbles when adding amplification mix to the slides.
  
• Remember to remove the SecureSeal chamber before the hexane wash.
  
• 20ul of amplification mix is sufficient for one slide. To much amplification mix may leads to large polonies.
  
3. Single base extension (SBE):

• The primer annealing protocol is: 94C for 6-min, then Tm-5 for 20-min. Optimal primer concentration is 1uM.
  
• Extension on the "A" allele often leads to non-specific fluorescent signals. These non-specific signals could no be removed by denaturation, suggesting that it may due to extension on dA at the anchored strain. A mock SBE with non-fluorescent dATP (and no primer) can effectively suppress the non-specific signals.
  
• Extension on the "G" allele is very difficult unless ThermoSequencase is used. We often type the "C" allele on the reverse strain instead.
  
• Both dideoxynucleotides or deoxynucleotides could be used for single base extensions. The advantage of using dideoxynucleotides is that one can potentially type all 4 allelles of two SNPs in one reaction. Besides, typing on the "G" allele gives better signals. However, fluorescent labelled dideoxynucleotides and Thermosequenase are more expensive than deoxynucleotides and DNA polomerase klenow fragment. It is very unfortunate that Applied Biosystem Inc. no longer sells AmpliTaq FS and Perkin Elmer removed many fluorescent labelled dideoxynucleotides from the catalog, which were used in one of our original protocols (Mitra et al. 2003). When using the deoxynucleotide protocol, only up to two alleles can be typed in one assay. This also depends on the downstream sequence of the SNP.  Here are a few typical cases (assuming dC is labelled by Cy3 and dT is labelled by Cy5):
• (C/T)ACA: This is the most simple case where both the C and the T alleles can be typed in one assay, because the adjacent nucleotide is "A". The extension reaction will stop right after the SNP variant site because there is no dATP or dGTP in the system.
• (C/T)CAA: In this case, the nucleotide adjacent to the SNP is "C", which means the Cy3 fluorophore will be incorporated no matter the template molecule is C allele or T allele at the variant site. As a result, "C" polonies will have only Cy3 signal while the "T" polonies will have both Cy3 and Cy5 signal. To avoid the crosstalking between the two alleles, one can do a first SBE with Cy3-dCTP only, wash the slide to remove all Cy3-dCTP, then perform the second SBE with Cy5-dUTP. The order need to be reversed if the adjacent nucleotide is "T". Please note that only one annealing reaction is necessary in this case.
• (C/T)CTG: This is more complicated that the second case, because, even with two independent SBE reactions, there will be crosstalking between the two channels. In such cases, two completely independent assays (including annealing, SBE, scanning, stripping off primer) are required for one SNP.
  
• A few tips on improving gel life. Do not use tissue paper or Kimwipes to wife off water from polony slides, because the gel can be accidentally damaged. Do a quick spin (500rpm 30 sec) with a centrifuge instead. Also scan the slides dry without coverslips. Gel can be damaged when you try to remove coverslip too. It is okay if the gel get dry out during scanning.