Biological sample preparation

introduction and background

Accessing biological information from nucleic acids and proteins is important for investigating a complex biological system. Consider the classic view of the central dogma of molecular biology, which is that coded genetic information from DNA is transcribed into messenger RNA (mRNA), and then proteins can be synthesized using the information in mRNA as a template. This basic construct suggests a full understanding of biological processes including aging, gene regulation, and phenotypic expression of mutant genes requires correlated genomic and proteomic studies. These studies should therefore benefit from purification and isolation of nucleic acids and proteins from the biological samples, particularly when the sample is precious and limited in volume.

In my Ph.D. research, I developed novel integrated sample preparation techniques to extract nucleic acids and proteins from complex biological samples using isotachophoresis (ITP). ITP is an electrophoretic technique that both separates and pre-concentrates ions based on their electrophoretic mobility. ITP is a robust sample preparation method and has been recently extensively applied to extraction and purification of both DNA and RNA targets from a variety of samples including blood, urine, and cell culture. ITP sample preparation of nucleic acids has also been shown to be compatible with downstream assays including qPCR and hybridization reactions.

Simultaneous Purification and Fractionation of Nucleic Acids and Proteins from Complex Samples Using Bidirectional Isotachophoresis

TOCI collaborated with Dr. Lewis Marshall and created an on-chip system to simultaneously purify and fractionate nucleic acids and proteins from complex samples using isotachophoresis (ITP). We have demonstrated this technique to simultaneously extract extracellular DNA and proteins from human blood serum samples and deliver these to two separate output reservoirs on a chip. The purified DNA is compatible with quantitative polymerase chain reaction (qPCR), and proteins can be extracted so as to exclude albumin, the most abundant protein in serum.

 

Bacterial RNA extraction and purification from whole human blood using isotachophoresis

I worked with Dr. Anita Rogacs and  developed a novel assay for physico-chemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with
Pseudomonas putida. This on-chip assay we developed is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from
degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays.
We showed that the purified RNA was compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR.

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