Automated Extraction of High-Molecular-Weight DNA

Introduction

Due to the steadily growing requirements placed on the samples to be processed, in academic environments as well as in the industry, automation solutions are taking on an ever increasing importance. Purification of genomic DNA (gDNA) is the starting point for a large number of subsequent processes where the quantity and purity of the isolated DNA is important. Conventional extraction methods use expensive reagents and are based on handling of small sample quantities as well as on the resulting purification procedures, which are repeated multiple times. In addition, this increases the risk of losing valuable sample material.

Application purpose

SmartExtraction is IST Innuscreen's innovative technology for extracting high-molecular-weight DNA. This technology forms the basis of a fundamentally new method for the automated isolation and purification of nucleic acids. SmartExtraction combines the patented extraction chemistry (DC Technology) with an intelligent surface, called "Smart Modified Surface". SmartExtraction significantly simplifies the entire automated workflow of DNA extraction and setting new standards with regard to efficiency, yield, and quality of the DNA. It is possible to obtain particularly large quantities of DNA. In addition, it is feasible to isolate high-molecular-weight DNA (200 kb - 500 kb) using an automated process. The "Smart Modified Surface" also facilitates mapping of the entire extraction process inside a pipette tip via simple pipetting steps.

The SmartExtraction technology does not require phenol/chloroform, ion exchangers, filter columns, of filter plates, nor is a suspension of magnetic or paramagnetic particles needed to bind the DNA. In addition, pre-filled and sealed reagent plastics make handling easier and can, in analogy to the functionalized pipette tips, be used directly on the CyBio FeliX pipetting system. For automated extraction of nucleic acids applying smartExtraction the CyBio FeliX can be used for 96 samples in parallel. Next to smart Blood DNA Midi prep additional protocols for different starting materials are available. This technology is particularly suited to meet the growing demand for automated handling of liquids.

CyBio FeliX is a flexible, fully automated pipetting system with 1 to 384 channels in the volume range form 1 to 1000 µL. In addition to the highly precise, parallel transfer in the 96- and 384-well format, pipetting can be also performed by single-channel, column, or row. The appropiate accessory is automatically determined and changed within a pipetting routine. CyBio FeliX combines highest flexibility with minimum space requirements, which is particularly reflected by its unique design with 12 positions on 2 levels. Owing to the modular concept of the CyBio FeliX, application specific configurations can be added at any time. On the basis of pre-configured and optimized pipetting routines, the focus always lies on the application.

Methods

Lysis of erythrocytes
3 mL of whole blood from each donor were used. Lysis of erythrocytes and pelleting of nucleated blood cells was performed in accordance with the information in the manual (Kit: smart Blood DNA Midi prep(a96)). After pelleting, the nucleated blood cells were completely resuspended in 120µL 1 x PBS.

Enzymatic lysis (proteolysis) and extraction
Proteolytic lysis of the cells was performed externally. To this end, 200µL of a lysis buffer (Lysis Solution CBV) and 30µL Proteinase K were added to the resuspended cells followed by incubation for 30 min on the BioShake iQ for 800 rpm at 55 ℃. Then the lysate was transferred into the reagent plate and placed onto device position 10 of the CyBio FeliX. Automatic processing and elution of the DNA samples was performed according to the information of the smart Blood DNA Midi prep(a96) protocol. The reagent plates of the kit required for automated DNA extraction are pre-filled with all reagents.

Verification of the DNA extraction
The extracted DNA was verified by using agarose gel electrophoresis. This involved applying 10µL of each eluate and 7µL of the DNA ladder to a 0,8% TBE gel (contains ethidium bromide, 5µL  of ethidium bromide for 100 mL of agarose gel). Electrophoretic separation took place in a horizontal gel electrophoresis system (Analytik Jena AG); by constantly applying of 127mA for optimal running conditions. The resulting DNA bands were visualized using UV light. The yield and the quality of the DNA were determined by using a spectrophotometer (ScanDrop®250, Analytik Jena AG). To this end, 4µL of each eluate were filled into an appropriate CHIPCUVETTE and measured by a path length of 1.0 mm.

Why you should choose this solution

SmartExtraction, a novel and innovative technology for isolating nucleic acids, can be elegantly and efficiently automated by using the flexible CyBio FeliX pipetting system for isolation of up to 96 samples in parallel. High-quality, reproducible pipetting results can be achieved, allowing the isolation of high yield of high-molecular-weight DNA. On the basis of a modular system, the degree of automation can be freely configured by the customer. The flexible automation solution contributes to improved reproducibility of the results and increases the efficiency of lab processes. Automatic extraction of DNA for downstream processes leads to simple, parallel preparation of samples with minimum erffort and maximum consistensy. In addition, the CyBio FeliX can also be used for other downstream routine liquid handling tasks.