PCR-generated padlock probes distinguish homologous chromosomes through quantitative fluorescence analysis

Abstract

Conventional cytogenetic techniques can distinguish homologous chromosomes in a qualitative manner based upon obvious morphological features or using in situ hybridization methods that yield qualitative data. We have developed a method for quantitative genotyping of single-nucleotide variants in situ using circularizable DNA probes, so-called padlock probes, targeting two different alpha satellite repeat variants present in human chromosome 7 centromeres, and a single-nucleotide variation in alpha satellite repeats on human chromosome 15 centromeres. By using these PCR-generated padlock probes, we could quantitatively distinguish homologous chromosomes and follow the transmission of the chromosomes by in situ analysis during three consecutive generations.

In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

“…These probes have detection specificity similar to that obtained with PCR [1][2][3] , but unlike in PCR, the reaction products can remain bound to their target sequences, even withstanding denaturing washes 4 . Unfortunately, detection signals from specifically circularized padlock probes have proven insufficient to reveal single-copy gene sequences owing to background noise from nonspecifically bound probes, although repeated centromeric sequences have been evaluated with sufficient precision to investigate the distribution of single-nucleotide variants in situ 5,6 .…”

From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

“…This probe is used for the localization of signals in in situ analyses. For example, a padlock probe was able to detect repeated alphoid sequences in metaphase chromosomes in a living cell, demonstrating its utility for in situ studies [ 53 – 55 ]. RCA is also known as a simple and efficient isothermal enzymatic process that utilizes strand-displacement DNA and RNA polymerases, like Phi29, Bst, and Vent exo-DNA polymerases for DNA and T7 RNA polymerase for RNA, to generate long single stranded DNAs and RNAs containing tens to hundreds of tandem repeats that are complementary to the circular template [ 56 – 58 ].…”

Fluorescence in situ hybridisation

“…Christian et al (2001) demonstrated that rolling circle amplification in situ can detect the gene copy number and single base mutations in fixed cells and can also detect and quantify the transcribed RNA in individual cells, making it a versatile tool for cell-based assays. Using the PCR-generated padlock probes, homologous chromosomes were quantitatively distinguished by Antson et al (2003), who observed the transmission of the chromosomes by the in situ analysis during three consecutive generations. According to Zhang et al (2006), the method of amplification of "padlock" probes (sometimes also called C-probes, i.e.…”