Polymerase extension at 3′ terminus of PNA-DNA chimera

Details Polymerase extension at 3′ terminus of PNA-DNA chimera Polymerase extension at 3′ terminus of PNA-DNA chimera

1999-08-13

2001-11-13

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091200, C536S022100, C536S023100, C536S024300, C536S024330, C536S025300, C536S025320, C536S025600

Reexamination Certificate

active

06316230

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to the fields of enzymology and nucleic acid analogs. Specifically, this invention is directed to template-dependent, primer extension of PNA-DNA chimera using polymerase enzyme and nucleotide 5′-triphosphates.
BACKGROUND
One of the most powerful and versatile tools available to molecular biologists is the in vitro replication of nucleic acid sequences by primer extension, as exemplified by the ubiquitous techniques of polymerase chain reaction (PCR) (Mullis, 1987) and DNA sequencing (Sanger, 1977). Both techniques include the steps of: 1) hybridizing a short, e.g. 15-30 nt, synthetic oligonucleotide primer to a single-stranded template nucleic acid; and 2) enzymatically extending from the 3′ hydroxyl terminus of the primer in the presence of nucleotide 5′-triphosphates, complementary to the template strand, and a polymerizing enzyme. By this general primer extension method, sequencing information is generated, template nucleic acids are amplified or copied, and other genetic analysis tests are conducted. Results are optimized through the choice and concentrations of primers, multiple primers, enzymes, nucleotides, and other reagents, and the selection of temperature, temperature cycling conditions, and other experimental conditions.
The choice of primers has been primarily limited to 2′-deoxyoligonucleotide primers made by the phosphoramidite chemistry method (Caruthers, 1983) on automated synthesizers (Caruthers, 1984). Whereas nucleic acid analogs are known which efficiently hybridize to DNA or RNA, some with comparable or superior hybridization specificity and/or affinity, enzyme-mediated formation of a new phosphodiester bond only occurs between a primer having a 3′ terminal hydroxyl and a nucleotide having a 5′-triphosphate, or a closely related isostere, i.e. &agr;-thiotriphosphate, etc. Most structural permutations in either the primer or the nucleotide severely compromise the efficiency of primer extension, or negate it totally.
Nucleic acid analogs are structural analogs of DNA and RNA and which are designed to hybridize to complementary nucleic acid sequences. Through modification of the internucleotide linkage, the sugar, and/or the nucleobase, nucleic acid analogs may attain any or all of the following desired properties: 1) optimized hybridization specificity or affinity, 2) nuclease resistance, 3) chemical stability, 4) solubility, 5) membrane-permeability, and 6) ease or low costs of synthesis and purification.
A useful and accessible class of nucleic acid analogs is the family of peptide nucleic acids (PNA) in which the sugar/phosphate backbone of DNA or RNA has been replaced with acyclic, achiral, and neutral polyamide linkages. The 2-aminoethylglycine polyamide linkage in particular has been well-studied and shown to impart exceptional hybridization specificity and affinity when nucleobases are attached to the linkage through an amide bond (Buchardt, 1992; Nielsen, 1991).
2-Aminoethylglycine PNA oligomers (
FIG. 1A
) typically have greater affinity, i.e. hybridization strength and duplex stability for their complementary PNA, DNA and RNA, as exemplified by higher thermal melting values (Tm), than the corresponding DNA sequences. The melting temperatures of PNA/DNA and PNA/RNA hybrids are much higher than corresponding DNA/DNA or DNA/RNA duplexes (generally 1° C. per bp) due to a lack of electrostatic repulsion in the PNA-containing duplexes. Also, unlike DNA/DNA duplexes, the Tm of PNA/DNA duplexes are largely independent of salt concentration. The 2-aminoethylglycine PNA oligomers also demonstrate a high degree of base-discrimination (specificity) in pairing with their complementary strand. Specificity of hybridization can be measured by comparing Tm values of duplexes having perfect Watson/Crick complementarity and those with one or more mismatches. The degree of destabilization of mismatches, measured by the decrease in Tm (&Dgr;Tm), is a measure of specificity. In addition to mismatches, specificity and affinity are affected by structural modifications, hybridization conditions, and other experimental parameters. The neutral backbone of PNA also increases the rate of hybridization significantly in assays where either the target, template, or the PNA probe is immobilized on a solid substrate. Without any electrostatic repulsion, the rate of hybridization is often much higher for PNA probes than for DNA or RNA probes in applications such as Southern blotting, northern blots, or in situ hybridization experiments (Corey, 1995). Unlike DNA, PNA can displace one strand, “strand invasion”, of a DNA/DNA duplex (Kuhn, 1999). With certain DNA sequences, a second PNA can further bind to form an unusually stable triple helix structure (PNA)
2
/DNA. PNA have been investigated as potential antisense agents, based on their sequence-specific inhibition of transcription and translation (Von Matt, 1999; Lee, 1998; Nielsen, 1996). PNA oligomers themselves are not substrates for polymerase as primers or templates, and do not conduct primer extension with nucleotides (Demers, 1997, see col. 2, lines 55-56).
PNA-DNA chimera are oligomer molecules with discrete PNA and nucleotide moieties. They can be synthesized by covalently linking PNA monomers and nucleotides in virtually any combination or sequence. Efficient and automated methods have been developed for synthesizing PNA-DNA chimera (Vinayak, 1997; Uhlmann, 1996; Van der Laan, 1997). PNA-DNA chimera are designed to have desirable properties found in PNA and DNA, e.g. superior hybridization properties of PNA and biological functions like DNA (Uhlmann, 1998).
Attempts to demonstrate primer extension of PNA-DNA chimeric primers with radioisotopically-labelled nucleotides have been reported. Primer extension on an 8 Int DNA template was attempted from a complementary PNA-DNA chimera with 15 PNA monomer units linked through an amide bond to a single 3′ terminal thymidine nucleoside (FIG.
1
B), various polymerases, and nucleotides dATP, dGTP, dTTP, and
32
P-dCTP (Lutz, 1998). Some incorporation of nucleotides and extension may be evident, but due to the unavailability of proper control experiments, the level of incorporation is unknown.
Primer extension was also reported using a mixture of PNA-DNA chimera consisting of 19 PNA monomer units with three (
FIG. 1D
) and four 2′-deoxynucleotides, labelled once and twice respectively, with
32
P dCTP and terminal transferase (Misra, 1998). The 3′ hydroxyl terminus was extended on a 49 nt DNA template and a 30 nt RNA template with unlabelled nucleotide 5′-triphosphates. Autoradiography of the gel after electrophoresis showed a ladder of radiolabelled products, the majority of which was unextended chimera, indicating inefficient primer extension. This experiment employed a relatively long PNA moiety, 19 monomer units, incurring the attendant costs, loss of specificity, and synthesis inefficiencies of a longer chimera oligomer.
In another study, chimera consisting of 3 PNA monomer units and either 2,4,6,9, or 12 deoxynucleotides were extended with Klenow polymerase from an 18 nt DNA template (Reeve, 1995). All chimera had T deoxynucleotide at the linkage between the PNA and DNA moieties. Detection of incorporated
32
P-dCTP by autoradiography indicated that all the chimera except the one with 2 deoxynucleotides were extended. However, no quantitative or qualitative data was provided. Given the sensitivity of autoradiography, extension of the chimera in this study may have been at a detectable, but not useful, level.
Fluorescence has largely supplanted radioactivity as the preferred detection method for most primer extension applications, such as automated DNA sequencing, in vitro DNA probe-based diagnostics, nucleic acid amplification, DNA fragment analysis, and transcriptional expression mapping and profiling. It is thus desirable to provide methods by which PNA-DNA chimera can be enzymatically extended to form non-radioisotopically labelled extension products. DNA sequ

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