John M. S. Bartlett.pdf - Bio-Nica.info

Arrays for Genotyping 267
4. Notes
1. The synthesis cells are produced by milling or moulding blocks of PTFE. The cavity is
generally 0.5- to 0.75-mm deep and the wall thickness 0.3 to 0.5 mm.
2. Polymerase-catalyzed extension requires the oligonucleotides to be attached through their
5′ ends. For ligation, the oligonucleotides on the array can be attached in either orientation,
using either the method described in Subheadings 2.1. or 2.2. Thus, deoxynucleotide
5′-dimethoxytrityl-3′- phosphoramidites replace the deoxynucleotide 3′-dimethoxytrityl-5′-
phosphoramidites. A requirement for ligation with oligonucleotides is that the 5′ end must be
phosphorylated. This can be achieved by the reaction of a chemical phosphorylating reagent
(Phosphate-On phosphoramidite available from most DNA synthesis reagent suppliers).
This is added as the last step in the chemical synthesis of the oligonucleotides. Phosphate
can also be added post synthetically using polynucleotide kinase and ATP (this reaction
works on both oligonucleotides in solution and array supported oligonucleotides).
3. The use of a linker between the surface and the oligonucleotides improves both hybridization
yield and access to the oligonucleotides by enzymes. Addition of phosphoramidites
based on polyethylene glycol (21) or addition of deoxythymidine phosphoramidites (22)
have both been used to add linkers before oligonucleotide synthesis.
4. Performing hybridizations and reactions of the array strips in puddles allows the volume
of liquid to be minimized. However, the chamber has to be made as humid as possible to
prevent evaporation, especially at elevated temperatures. The methods described use Petri
dishes with wet tissues inside, but the tissues can also be draped over the top of the dish.
The Petri dishes can be placed in sealed plastic containers. Sealed tubes may also be used
and although the volumes are often greater than in puddles of solution, they can be more
easily incubated in hot blocks or in water baths.
Both the hybridization and washing buffer and temperature can be varied. Conditions
should be found that give high yields of duplex and good discrimination between matched
and mismatched duplexes.
5. For hybridizations below 37°C, care must be taken not to warm the plates by touching
with fingers because this can cause the melting of short duplexes. For hybridizations below
room temperature, all the apparatus that comes in contact with the array must be cooled to
the hybridization temperature or below.
6. When using large RNA targets, secondary and tertiary structures can become a problem
preventing hybridization. One method to alleviate this is to randomly cleave the RNA
in base.
7. For assays, including reactions with enzymes, both the buffer and temperature of the
hybridization reaction can be varied. Using a two-step hybridization followed by reaction
procedure the hybridization buffer need not be the buffer required for the enzymatic
reaction. Also, because many of the enzymes used do require specific temperatures, using a
two-step hybridization/reaction the hybridization temperature can be completely different
from the enzyme-catalyzed reaction.
8. The reporter oligonucleotide is typically 5′ end labeled with a radio label using standard
methods. If fluorescence labeling is used, the label is incorporated during oligonucleotide
synthesis.
9. In our methods, radiolabels were used. These are convenient for many applications because
they are readily commercially available and are easily detected using a phosphorimager.
Other label types can be used and in most cases there are similar ready-labeled reagents
available. We have used Cy5 labels on oligonucleotides in ligation assays and nucleotide
triphosphates in polymerase extension assays. This label can be detected using a STORM
phosphorimager. Because of the large fluorescent background of the polypropylene support
at short wavelengths, we have found that fluorescein is not useful with polypropylene.