PNAs for Genetic Analysis Using MALDI-TOF MS,Polymorphisms,Magnetic Beads,Non Biotinylated Strand,PNA Probes,Desorption,Ionization,Hybridization

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Triplex DNA, TFOs, PNAs, RNA-DNA Hybrids and dsRNA - RNAi Triplex DNA, TFOs, PNAs, RNA-DNA Hybrids and dsRNA - RNAi - Introduction Triplex DNA and TFOs H Form or H DNA PNAs Peptide Nucleic Acids PNA DNA and PNA RNA Hybrid Complexes Synthesis of PNAs by BOC or Fmoc Protocols Modification of PNAs for Specific Uses

Home >> Biotechnology and Genomics >> Triplex DNA, TFOs, PNAs, RNA-DNA Hybrids and dsRNA - RNAi >> PNAs for Genetic Analysis using MALDI-TOF MS

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PNAs for genetic analysis using MALDI-TOF MS.
PNAs can also be used as allele specific probes for hybridization with PCR amplified DNA, for detection of multiple, single base polymorphisms. Following steps are involved.
(i) immobilization of biotinylated target DNA (e.g. PCR product), using Streptavidin-coated magnetic beads;
(ii) dissociation and removal of the non-biotinylated strand;
(iii) hybridization with mass labelled PNA probes;
(iv) washing to achieve proper discrimination and
(v) direct analysis of hybridized PNAs using MALDI-TOF MS.

During MALDI (matrix-asociated laser desorption/ionizations), the PNA probes hybridized to the immobilized DNA target, are dissociated and desorbed from the immobilized target strand, enabling their detection by MALDI-TOF MA. PNAs offer following advantages over conventional DNA probes:
(i) increased hybridization, stability and specificity,
(ii) ability to hybridize at low-salt conditions.

Different steps involved in Hybridization of PNA, used as an allele specific probe for detection of multiple single base polymorphisms, by MALDI-TOF MS.

Different Steps involved in Hybridization of PNA, used as an allele specific probe for detection of multiple single base polymorphisms, by MALDI-TOF MS.

The above strategy was used for detecting mutations at four polymorphic sites in exon 4 of the human tyrosinase gene, that is implicated in albinism. Two PNA probes were designed for each polymorphic site, one for wild allele and the other for mutant allele. The two probes were uniquely mass-labelled (Lm, Ln each giving a distinct peak, when analysed by MALDI-TOF MA). The mass labels were 8-amino-3, 6-dioxaoctanoic acid molecules, each with a molecular weight 146 Da. The labels are easily attached during synthesis of PNA molecule.

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