High-specificity single-tube multiplex genotyping using Ribo-PAP PCR, tag primers, alkali cleavage of RNA/DNA chimeras and MALDI-TOF MS.

Here, we describe a high-throughput, single-tube, allele-specific ribonucleotide analog pyrophosphorolysis-activated polymerization (ribo-PAP) PCR multiplex genotyping and resequencing method. An RNA/DNA chimeric PCR product is generated using genomic DNA as starting template, a panel of allele-selective 5'-tagged primers, a reverse primer, one nucleotide in the ribo-form (90-100%), the other nucleotides in the deoxy-form, a DNA polymerase capable of incorporating ribonucleotides, a suitable buffer and thermal cycling. The RNA/DNA chimeric PCR products are fragmented by treatment with alkali and analyzed by mass spectrometry. All allele-selective primers have a 5' repetitive motif where each repeat unit has a unique, distinct mass upon reverse copying and alkali fragmentation. The mass of the complement repeat fragment or flag identifies the primer or primers that were recruited in the ribo-PAP PCR. The method readily identifies homozygous and heterozygous positions in simplex or duplex ribo-PAP PCR. Many different tags can be analyzed simultaneously. The assay can genotype several SNPs in a single tube. It thus constitutes the simplest genotyping protocol with multiplex analysis. This novel genotyping and resequencing protocol was applied to different genomic loci: NOS1 and H19 in 30 individuals in simplex ribo-PAP PCR and at two SLCO1B1 loci in 95 individuals in duplex ribo-PAP PCR.

Ribo-polymerase chain reaction--a facile method for the preparation of chimeric RNA/DNA applied to DNA sequencing.

We describe ribo-polymerase chain reaction (PCR), a method for the preparation of chimeric RNA/DNA. The RNA/DNA chimeric nucleic acids are generated directly from genomic DNA starting templates with two locus-specific primers, three nucleotides in their deoxy form and the fourth in its ribo form, a DNA polymerase capable of incorporating ribo bases, a suitable buffer, and thermal cycling. We have applied ribo-PCR to resequence DNA by directly fragmenting the RNA/DNA chimeras with alkali and analyzing the fragments by mass spectrometry (MS). Mass fingerprint is used to identify deviations from the reference sequence. This method readily detects homozygous sequence deviations as well as heterozygous positions directly from genomic DNA samples. With the high-throughput capability of MS, this facile method is well suited for screening DNA sequences of limited regions of the genome in a large number of individuals. It can also be used to sequence multiple distant genomic loci in a single reaction. This novel ribo-PCR resequencing protocol was applied to different genomic loci involving nitric oxide synthase 1 (NOS1) and H19 in 30 individuals and SLCO1B1 in 95 individuals.

DNA sequencing by MALDI-TOF MS using alkali cleavage of RNA/DNA chimeras.

Approaches developed for sequencing DNA with detection by mass spectrometry use strategies that deviate from the Sanger-type methods. Procedures demonstrated so far used the sequence specificity of RNA endonucleases, as unfortunately equivalent enzymes for DNA do not exist and therefore require transcription of DNA into RNA prior to fragmentation. We have developed a novel, rapid and accurate concept for DNA sequencing using mass spectrometry and RNA/DNA chimeras and applied it to sequence mitochondrial DNA. Our method is based on the preparation of a chimeric RNA/DNA with a DNA polymerase that also incorporates ribonucleotides. Sequencing is carried out with one ribonucleotide (ATP, CTP or GTP) and the other three nucleotides in their deoxyribo-form. The product is treated with alkali, which cleaves 3' of all ribonucleotides to form a terminal 3' phosphate. Conditions have been streamlined so that molecular, biological and alkali cleavage conditions are compatible with matrix-assisted laser desorption/ionization time-of-flight (MALDI) mass spectrometric analysis. Fragment analysis by MALDI MS provides a sequence-specific fingerprint, which allows the identification of differences between a reference and another sequence. Due to the mass profile, the position and kind of the mutation can be assigned. These differences between signatures are indicative of known, unidentified, rare and private mutations. This novel DNA sequencing protocol was applied to sequence the hypervariable region 1 (HV1) of mitochondrial DNA in 22 individuals.

SNP genotyping using alkali cleavage of RNA/DNA chimeras and MALDI time-of-flight mass spectrometry.

Single nucleotide polymorphisms (SNPs) are now widely used for many DNA analysis applications such as linkage disequilibrium mapping, pharmacogenomics and traceability. Many methods for SNP genotyping exist with diverse strategies for allele-distinction. Mass spectrometers are used most commonly in conjunction with primer extension procedures with allele-specific termination. Here we present a novel concept for allele-preparation for SNP genotyping. Primer extension is carried out with an extension primer positioned immediately upstream of the SNP that is to be genotyped, a complete set of four ribonucleotides and a ribonucleotide incorporating DNA polymerase. The allele-extension products are then treated with alkali, which results in the cleavage immediately after the first added ribonucleotide. In addition, to obtain fragments easily detectable by mass spectrometry, we have included a ribonucleotide in the primer usually at the fourth nucleotide from the 3' terminus. The method was tested on four SNPs each with a different combination of nucleotides. The advantage over other mass spectrometry-based SNP genotyping assays is that this one only requires a PCR, a primer extension reaction with a universal extension mix and an inexpensive facile cleavage reaction, which makes it overall very cost effective and easy in handling.

Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry.

In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or alpha-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and alpha-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease.

Chimeric thermostable DNA polymerases with reverse transcriptase and attenuated 3'-5' exonuclease activity.

The synthesis of accurate, full-length cDNA from low-abundance RNA and the subsequent PCR amplification under conditions which provide amplicon that contains minimal mutations remain a difficult molecular biological process. Many of the challenges associated with performing sensitive, long RT/PCR have been alleviated by using a mixture of DNA polymerases. These mixtures have typically contained a DNA polymerase devoid of 3'-5' exonuclease, or "proofreading", activity blended with a small amount of an Archaea DNA polymerase possessing 3'-5' exonuclease activity, since reverse transcriptases lack 3'-5' exonuclease activity and generally have low fidelity. To create a DNA polymerase with efficient reverse transcriptase and 3'-5' exonuclease activity, a family of mutant DNA polymerases with a range of attenuated 3'-5' exonuclease activities was constructed from a chimeric DNA polymerase derived from Thermus species Z05 and Thermotoga maritima DNA polymerases. These "designer" DNA polymerases were fashioned using structure-based tools to identify amino acid residues involved in the substrate-binding site of the exonuclease domain of a thermostable DNA polymerase. Mutation of some of these residues resulted in proteins in which DNA polymerase activity was unaffected, while proofreading activity ranged from 60% of the wild-type level to undetectable levels. Kinetic characterization of the exonuclease activity indicated that the mutations affected catalysis much more than binding. On the basis of their specificity constants (kcat/KM), the mutant enzymes have a 5-15-fold stronger preference for a double-stranded mismatched substrate over a single-stranded substrate than the wild-type DNA polymerase, a desirable attribute for RT/PCR. The utility of these enzymes was evaluated in a RT/PCR assay to generate a 1.7 kb amplicon from HIV-1 RNA.