Reverse transcription-polymerase chain reaction on a microarray: the integrating concept of “active arrays”

Abstract

In this report we describe the proof of principle of a reverse transcription polymerase chain reaction (RT-PCR) but on-chip, with immobilized specific primers using a transcriptome of mouse-muscle fibroblasts for detection of muscle-specific expression products of these cells. The isolated total mRNA was directly incubated on an array of immobilized and solubilized specific primers, which allow the amplification of certain muscle-specific RNAs via its immobilized cDNAs. In contrast to others, the immobilized cDNA-products were directly synthesized on the chip by applying covalently bound specific primers. The products were detected by the incorporated and fluorophore-modified specific primers of the subsequently synthezised second strand. In addition, this second-strand served as a further template (like the basically used mRNA) in the subsequent solid-phase-PCR to amplify first-strand cDNA copies at the remaining immobilized specific primer-probes. This is the intrinsic factor of the amplification of certain signals of this application. The specific cDNA templates of genes coding for subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm), and ATPase (Atp2a2) were amplified on a biochip by RT-PCR directly from freshly isolated mRNA. The resulting procedure allows the detection of mRNA sequences from less than 5 pg of total RNA preparations.

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…In their work there appeared to be no significant difference in the gene expression performance whether a two-(ImPromII reverse transcriptase for reverse transcription, and Taq DNA polymerase for cDNA amplification) or a oneenzyme (rTth DNA polymerase for both reverse transcription and cDNA amplification) amplification system was employed [2].…”

“…Accordingly, there has been a plethora of applications on its implementation on microarrays for qualitative and quantitative nucleic acids analysis [23,[28][29][30][31], species discrimination [23,32,33], point mutations and single nucleotide polymorphs (SNPs) [34][35][36][37] and gene expression analysis [2,38]. The design of the approach is very straightforward whereby a PCR primer is covalently tethered to a solid-phase support, while the remaining components of the PCR mixture which include an opposite primer, DNA polymerase, buffer constituents and target DNA are present in the solution covering the microarray.…”

“…Von Nickisch-Rosenegk et al [2] have also shown a reverse transcription reaction on a microarray immobilized with a primer followed by subsequent solid-phase PCR for gene expression analysis. In their work there appeared to be no significant difference in the gene expression performance whether a two-(ImPromII reverse transcriptase for reverse transcription, and Taq DNA polymerase for cDNA amplification) or a oneenzyme (rTth DNA polymerase for both reverse transcription and cDNA amplification) amplification system was employed [2].…”

“…More recently attention has been drawn to the use of "active arrays" [1,2] which offer the possibility of performing different enzymatic reactions with immobilized biomolecules on a solid support, thus providing additional sensitivity, specificity and scalability of analysis. The most successful implementation of active arrays has been in the development of high throughout DNA sequencing (HTS), also known as next generation sequencing (NGS).…”

A Light‐Activated DNA Polymerase

“…Beyond this temporal control of DNA polymerization, light activation also offers the possibility of spatial control of locally induced polymerase activity to facilitate the design of novel nanodevices and biotechnological applications, such as on-chip real-time PCR detection. [18] Experimental Section Gene manipulation, bacterial culture, and protein purification: The E. coli strain BL21 was transformed with the plasmid pTaq and used to express wild-type Taq polymerase induced with 0.5 mm IPTG. All primers were synthesized by IDT DNA.…”