Padlock probes : Circularizing oligonucleotides for localized detection of DNA sequence variants

• Present techniques for localized detection of DNA sequence variants have limitations due to problems with sensitivity, specificity, signal localization or ability to perform many analyses simultaneously.In this study a novel gene analytic reagent is presented - the padlock probe. Padlock probes are linear oligonucleotides with target complementary sequences at both ends, joined by a non-target complementary segment. Upon hybridization to a target DNA strand, the end-sequences are brought together and can be joined by a DNA ligase. Thereby the probe becomes circularized and threaded on the target DNA strand. Ligated probes were shown to be circular and topologically linked to circular target molecules. Thetopological link formed between circularized padlock probes and target DNA strands was shown to resist denaturing washes.A novel procedure for oligonucleotide synthesis was developed that allows for cleavage and removal of depurinated products before oligonucleotides are released from the synthesis support. In this manner molecules with intact ends can be isolated after an ordinary HPLC purification. The novel procedure is of general utility in oligonucleotide synthesis and especially suitable for syntheses of terapeutic antisense reagents and of padlock probes.Padlock probes were used to distinguish a single nucleotide difference in situ in metaphase chromosomes. Two alpha-satellite sequences present in the centromeres of human chromosomes 13 and 21 were selectively identified by two probes labelled in distinct colors. The two repeat sequences were shown to be polymorphic among different homologues of chromosomes 13 and 21, allowing identification of the parental origin of each of these chromosomes in an individual.For enhanced detection circularized padlock probes can be used to template a rolling circle replication reaction, providing a linear signal amplification suitable for quantitative analyses. The highly processive Φ 29 DNA polymerase was superior to the Kleenow DNA polymerase in initiating rolling circle replication by displacing the circular padlock probes from target DNA strands. Efficient amplification requires release of probes from target DNA strands. This is accomplished when displaced probes slide off a nearby end of the target molecule.The combined properties of the padlock probes render them promising reagents for future DNA diagnostics.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Medicinsk genetik (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Medical Genetics (hsv//eng)

Nyckelord

Genetics

Genetik

Clinical genetics

Klinisk genetik

Medical Genetics

medicinsk genetik

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