Biomedical subjects

M Ronaghi

Publications and source records attributed to M Ronaghi.

22 records · Page 2Linked to original sources

PubMed1998

PCR-introduced loop structure as primer in DNA sequencing.

The need for a primer hybridization step before sequencing has been eliminated using a stem-loop structure generated by PCR. The loop structure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capture of the PCR product, the loop is formed by elution of the non-bound strand. Here, we show that both the immobilized and the eluted strand can be analyzed using conventional Sanger DNA sequencing and the novel pyrosequencing method as described previously. By using a stem-loop structure as a primer for DNA sequencing, the risk for mispriming is minimized.

Base Sequence↗

PubMed1997

Detection of single-base changes using a bioluminometric primer extension assay.

A rapid bioluminometric technique for real-time detection of known single-base changes is presented. The concept relies on the measurement of the difference in primer extension efficiency by a DNA polymerase of a matched over a mismatched 3' terminal. The rate of the DNA polymerase-catalyzed primer extension is measured by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (P. Nyrén (1987) Anal. Biochem. 167, 235-238). The PPi formed in the polymerization reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the single-base detection assay, immobilized single-stranded DNA fragments are used as template. Two detection primers differing with one base at the 3' end are designed, one precisely complementary to the nonmutated DNA sequence and the other precisely complementary to the mutated DNA sequence. The primers are hybridized with the 3'-termini over the base of interest and the primer extension rates are, after incubation with DNA polymerase and deoxynucleotides, measured with the ELIDA. We show that the relative mismatch extension efficiency is strongly decreased by substituting the alpha-thiotriphosphate analog for the next correct natural deoxynucleotide after the 3'-mismatch termini. The possibility of using the technique for studies of mismatch extension kinetics for two polymerases lacking exonucleolytic activity is shown.

Biological Assay↗

PubMed1996

Real-time DNA sequencing using detection of pyrophosphate release.

An approach for real-time DNA sequencing without the need for electrophoresis has been developed. The approach relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (Nyrén, P. (1987) Anal. Biochem. 167, 235-238). The PPi formed in the DNA polymerase reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the sequencing procedure, immobilized single-stranded template was used in a repeated cycle of deoxynucleotide extension. Real-time signals in the ELIDA, proportional to the amount of incorporated nucleotide, were observed when complementary bases were incorporated. An increased signal-to-noise ratio was obtained by substitution of deoxyadenosine alpha-thiotriphosphate (dATP alpha S) for the natural deoxyadenosine triphosphate, dATP alpha S is efficiently used by the DNA polymerase, but is not recognized by the luciferase. As a model, 15 bases of a single-stranded PCR product were sequenced. The possibility for parallel processing of many samples in an automated manner is discussed.

Adenosine Triphosphate↗

PubMed1996

Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons.

The so-called Mycoplasma mycoides cluster consists of six species or subspecies of mycoplasmas (Mollicutes). These species are pathogenic for ruminants and some of them are of great concern in veterinary medicine. The members of the M. mycoides cluster have two rRNA operons (rrnA and rrnB). The nucleotide sequences of the 16S rRNA genes of 10 strains, representing all of the known species and subspecies of the M. mycoides cluster, were determined by direct automated solid-phase DNA sequencing. The sequences of both rRNA operons were determined by a novel strategy involving in vitro amplification by PCR with one operon-specific primer pair and one general primer pair. Interestingly, sequence differences (polymorphisms) between the two operons were observed for all strains. Two strains of M. capricolum subsp. capripneumoniae were sequenced, and 15 polymorphisms were found in the type strain (F38) and 17 polymorphisms were found in the other strain (4/2LC). Eight polymorphisms were found in the 16S rRNA genes of the M. mycoides subsp. mycoides small-colony type, and sequence length variations in a poly(A) region were observed in the 16S rRNA genes of the two operons of this species. Secondary-structure analysis showed that polymorphisms were present in both stem and loop regions. The nucleotide substitutions in the polymorphic sites of the stem regions often resulted in a change from a canonical to a noncanonical base pairing or vice versa. A compensatory mutation was never observed in the other nucleotide of the base pair. Phylogenetic analysis based on the 16S rRNA sequences indicated that Mycoplasma sp. strain PG50 should be included in the M. capricolum species group. Furthermore, the 16S rRNA sequences of M. mycoides subsp. capri and the M. mycoides subsp. mycoides large-colony type were 99.9% identical. We therefore suggest that these species be reclassified in a common species group (for instance, "Mycoplasma capri") distinct from the M. mycoides subsp. mycoides small-colony type, which formed an intermediate branch between the M. capricolum species group and the M. capri species group.

Base Sequence↗