Thermal cycler is used for polymerase chain (PCR) amplification of the template DNA to generate numerous million DNA copies in a limited time of ~2-3 hours. Gradient Thermal Cycler is used for determination of an optimal annealing temperature in a lesser time using the least number of PCR programming steps. BioRad’s Gradient Thermal Cycler allows up to 10-12 different annealing temperature gradient set up in one single run. It also allows the amplification of a maximum of 96 samples in a single run. The Thermal Cycler also enables the reverse transcriptase PCR and other PCR variants.
MLPA is a multiplex PCR assay that is used to determine the relative copy number (deletions and duplications) of several DNA sequences for a specific disorder or syndrome with respect to the reference sample. This assay utilizes up to 40-50 different probes, each specific for different DNA sequences of a particular phenotype. MLPA reaction constitute five main steps: i) denaturation and probe hybridization, ii) probe ligation, iii) PCR amplification with fluorescent primers, iv) capillary electrophoresis and v) data analysis. MLPA assay differs from conventional PCR amplification of a DNA sequence as it involves the amplification of ligated probe regions rather than the target DNA.
Nanopore MinION sequencing technology enables single molecule sequencing via a direct and real-time analysis of long DNA or RNA fragments. Nanopore technology works by the detection of changes in the ionic or electric current as nucleic acids are passed through a biological membrane or a nanopore. The generated ionic current signals are further decoded to provide the bases of the DNA or RNA sequence. This long-read sequencing technology is utilized in resolving complex genomic structural variants and repetitive regions, detection of the epigenetic modifications, de novo genome assembly, improving the existing reference genome, sequencing of entire microbial genome in single reads, and metagenomic identification of closely related species.
QIAGEN's real-time PCR cycler, the Rotor-Gene Q, combines multiple optimized design features to provide the outstanding performance and reliable results for our research demands. Together with optimized QIAGEN kits for real-time PCR, the Rotor-Gene Q enables streamlined analysis for a wide range of applications, such as, gene expression analysis, pathogen detection, DNA methylation analysis, genotyping and gene scanning, and miRNA research.
Agilent whole human genome oligonucleotide arrays were used for any transcriptomic analysis carried out at IOB. A 60-mer oligonucleotide probe represents each spot in an array. Each array contains 44k spots representing 41,000 genes. A high density array, 4X44K was used for simultaneous analysis of 4 samples.
High resolution whole human genome array from Agilent was used to analyze copy number variations. High throughput analyses were carried out using high density arrays 4X44K or 8X60K, where 4 or 8 samples could be analyzed simultaneously. Comprehensive probe coverage spans coding and non-coding regions with emphasis on known genes, promoters, miRNAs, disease, pseudoautosomal, and telomeric regions.
IOB has designed human X-chromosome tiling microarrays using Agilent’s e-array, a web portal for designing custom arrays. The custom designed array has 244,000 probes covering human X-chromosome with an average spacing of 635 bp that provides high resolution. The array encompasses unique probes that represent most exons of the X-chromosome genes. These arrays will be able to detect micro-duplications and micro-deletions of size less than 1 Kb. These custom designed high resolution X-chromosome tiling arrays will be used to study the copy number variations in X-linked mental retardation.
Use of tissue microarrays and cell pellet arrays provides us another high throughput platform to validate and test numerous potential biomarkers which have been identified using high resolution genomic and proteomic technologies. Advantage of using these microarrays is that we can validate a potential molecular signature in cancer or any other context against hundreds of tissue samples or cell lines belonging to different pTNM, age group, gender and population. This will also provide confidence and statistical proof for the biomarkers identified from otherwise low number of samples used for DNA microarrays and mass spectrometry approaches in the biomarker discovery phase.
