ABSTRACT
Base-matching or so-called mini-sequencing is a powerful technique for genotyping and mutation
identification. However, its application is often hampered by high background and high cost. We have decreased the background by
~
5-fold by incorporating an end-blocking step and using only 1/10 of the usual nucleotide
concentrations.
Precise determination of a genotype or identification of a point mutation is
often carried out by an assay in which the interrogated base is revealed after
it is matched with a known complementary dideoxynucleotide. This single-base primer extension, or base- matching, is referred to as mini-sequencing or primer guide- nucleotide incorporation (
1
,
2
). The assay uses the polymerase chain reaction (PCR) (
3
) for generating template molecules in a sufficient number and the Sanger
sequencing reaction (
4
) for incorporating a single complementary fluorescent dideoxynucleotide into a template-probe hybrid.
In a typical base-matching assay (
5
), single-stranded and biotin-labeled molecules of template DNA generated by PCR are first immobilized on a streptavidin-coated solid support. A specific oligonucleotide (probe) is then hybridized to the immobilized DNA. The base in the template DNA that immediately follows the 3'-end of the probe is then identified after
extending the probe sequence with a complementary dye-conjugated dideoxynucleotide. However, the signal is often confounded by a high background that is
likely to have resulted from non-specific extension at the 3'-end of the biotinylated sequences involved in secondary
structures, or from extension of probe molecules in non-specific sites. Our modified procedure, which incorporates a blocking step
and low nucleotide concentrations, solves the background problem. We describe the refined procedure and its application in detection of mutations
related to sickle cell disease.
The procedure that we have been using is as follows:
1. Perform PCR with a 5' biotin-labeled primer and a normal 3' primer under standard conditions. A clear band should be
seen by analyzing 5 [mu]l of the PCR product on an agarose gel. Dilute an aliquot of the amplified
DNA 1:50 in Binding Buffer (5* SSC, 8% formamide, 8% Triton X-100, 1 mg/ml BSA).
2. Wash a streptavidin-coated well (either low or high capacity in an 8-well strip; Boehringer Mannheim) once with the Binding Buffer; add
20 [mu]l of the diluted sample to the well; incubate the solution at 37[iexcl]C for 15 min.
3. Discard the solution, then add 100 [mu]l Denaturing Solution (0.1 M NaOH, 1 mM EDTA) to the well for 5 min at room temperature.
4. Wash the well 2 times with Washing Buffer (PBS + 0.05% Tween 20), once with
Extension Buffer
(25 mM MOPS, pH 7.5, 50 mM NaCl; 10 mM Na
3
citrate, 5 mM MnCl
2
, 5 mM DTT, 1 mg/ml BSA).
5. Add 20 [mu]l Blocking Solution which consists of Extension Buffer plus a mixture of the four dideoxynucleotides each at 1 [mu]M (ddNTP, N: a mixture of different nucleotides) and 0.4 U Sequenase (USB). Incubate the solution at room temperature for 15 min. Wash the
well once with the Extension Buffer.
6. Add 20 [mu]l Labeling Solution, which consists of Extension Buffer plus 0.5 [mu]M of a specific primer (probe), 0.25 [mu]M of a required fluorescein conjugated dideoxynucleotide (F-ddXTP, X: one of the four nucleotides; DuPont NEN), a mixture of ddNTP
(minus ddXTP) each at 1 [mu]M, and 0.4 U Sequenase. Incubate the reaction at 37oC for 15 min.
7. Wash the well 3 times with the Washing Buffer. Obtain results by measuring fluorescence emissions. Alternatively, add peroxidase-conjugated anti-F at a dilution recommended by its manufacturer (i.e. 1:2000 in PBS containing 5 mg/ml BSA). Incubate the reaction at 37oC for ~15 min; wash the well 5 times with the Washing Buffer; perform color reactions (5-15 min) following the manufacturer's instructions.
The introduction of a blocking step (Step 5) and the use of low concentrations of nucleotides are designed to reduce background. To demonstrate the effects of these modifications, we assayed DNA obtained from
the normal human adult beta-globin gene sequence (A-gene) and the S variant gene sequence (S-gene) (
6
,
7
). Independent amplifications were performed on each of these DNAs using the same
primer set (5GNB: Biotin 5'-tgt gga gcc aca ccc tag ggt tg-3' and 3CBM: 5'-ctt gcc atg agc ctt cac att agg-3'). Probe AC (5'-ggc agt aac ggc
aga ctt ctc ct-3') and F-ddCTP were used to interrogate for the first base, G, of the
6th codon in the A-gene (A-codon). Probe ACS (5'-ggc agt aac ggc aga ctt ctc c-3') and F-ddATP were used to interrogate for
the second base, T, of the 6th codon in the S-gene (S-codon). A positive test, defined as an assay in which the A-gene sequence was assayed for `A-codon' or the S-gene sequence for `S-codon', was expected to produce a higher O.D. reading than when, for
example, the A gene sequence was interrogated for the presence of the S-codon (negative test). Therefore, the O.D. reading derived from a negative
test defined the background value for the corresponding positive test.
Figure
1
shows that when the blocking step was incorporated into the published protocol
(
5
), the background dropped 2-3-fold. Lowering nucleotide concentrations to 1/10 of the published
concentrations (
5
) reduced the background further. Using both modifications, we were able to decrease the background significantly, resulting in a 5-fold increase in the signal/background ratio. Relatively higher
backgrounds in positive tests for A-gene (as compared to positive tests for S-gene) suggest that other factors may contribute, but the present modifications diminish the backgrounds to an acceptably low level.
*To whom correspondence should be addressed. Tel: +1 617 983 6351; Fax: +1 617
522 2846; Email: xsu@world.std.com
REFERENCES
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