Jan Berka

Separation of DNA fragments by capillary electrophoresis using replaceable linear polyacrylamide matrices.

The use of low percent (1.5-6% T) replaceable linear polyacrylamide (LPA) network matrices for rapid separation of double-stranded DNA fragments was explored. Separations of fragments ranging from 20 to 23,000 base pairs were readily achieved. Typically, 4 x 10(6) theoretical plates/m were obtained in less than 30 min. Short separation times under 2 min were also possible, using the DNA intercalating dye, ethidium bromide, along with high electric fields. The high resolving power of linear polyacrylamide was demonstrated in the separation of two fragments which differ by a single base pair (123/124 base pairs) using 6% T LPA and ethidium bromide intercalation. This LPA composition allowed for the possible single base-pair resolution of dsDNA fragments up to 300 base pairs in length. Several concentrations of the linear polyacrylamide for different ranges of fragment lengths have been employed. In addition, replaceable LPA offers the advantage of a fresh separation matrix for each run, thus overcoming column stability problems and minimizing needs for sample cleanup. Electro-osmotic flow was substantially reduced using stable capillary coatings, which were required for obtaining high efficiencies and good reproducibility.

A transmission imaging spectrograph and microfabricated channel system for DNA analysis.

In this paper we present the development of a DNA analysis system using a microfabricated channel device and a novel transmission imaging spectrograph which can be efficiently incorporated into a high throughput genomics facility for both sizing and sequencing of DNA fragments. The device contains 48 channels etched on a glass substrate. The channels are sealed with a flat glass plate which also provides a series of apertures for sample loading and contact with buffer reservoirs. Samples can be easily loaded in volumes up to 640 nL without band broadening because of an efficient electrokinetic stacking at the electrophoresis channel entrance. The system uses a dual laser excitation source and a highly sensitive charge‐coupled device (CCD) detector allowing for simultaneous detection of many fluorescent dyes. The sieving matrices for the separation of single‐stranded DNA fragments are polymerized in situ in denaturing buffer systems. Examples of separation of single‐stranded DNA fragments up to 500 bases in length are shown, including accurate sizing of GeneCalling® fragments, and sequencing samples prepared with a reduced amount of dye terminators. An increase in sample throughput has been achieved by color multiplexing.

Capillary electrophoresis with polymer matrices: DNA and protein separation and analysis.

Publisher Summary This chapter discusses various approaches to the size separation of DNA and sodium dodecyl sulfate (SDS)-protein complexes, by capillary electrophoresis. It first briefly discusses cross-linked gel-filled capillary columns, followed by noncross-linked polymer network-based columns, the latter being in predominant use today, especially in a replaceable format. In this latter case, the polymer solution is removed from the capillary tube, by pressure, and fresh polymer solution is reloaded into the column after each analysis. A survey of the typical matrices that have been successfully employed as well as some practical details on their implementation is discussed in the chapter. Fortunately, many commercial vendors have kits available to conduct such separations and these kits can often be directly used in commercial equipment. This discussion then turns to providing specific examples and practical details of the application of such systems to DNA and protein analysis. The breadth of the applications of capillary electrophoresis (CE), with polymer matrices, has been demonstrated in this chapter. The use of replaceable matrices is critical to long-term column usage in an automated fashion. The type, molecular weight (MW), and concentration of the polymer play an important role in the separation achieved. Capillary electrophoresis is predicted to become an important tool for DNA sequencing, sizing, and mutation analysis in the forthcoming years.

DNA sequence analysis of Prinker-modified restriction fragments after collection from capillary electrophoresis with replaceable matrices.

This paper demonstrates the procedure of sequencing DNA restriction fragments isolated by a recently developed fraction collector after CE separation. In particular, using pBr 322 plasmid as a model system, a double digest was performed with Eco RI and Pst 1 restriction enzymes to produce two fragments of 749 base pairs (bp) and 3612 bp, both with cohesive ends. Prinkers, specific linkers complementary to the cohesive ends, were then ligated to both fragments (increasing the size by 59 bp each). These Prinker-modified fragments were separated by CE and collected. The success of the collection was demonstrated by reinjection of each isolated fraction with laser-induced fluorescence detection, using ethidium bromide as intercalater. The 808 bp isolated fragment was then polymerase chain reaction-amplified with appropriate primers for the Prinker ends, followed by cycle sequencing. Both strands of the fragment were run on an ABI 373, sequencing 427 bases and 450 bases, respectively, with a read accuracy of 99.3%. This approach with Prinker-modified restriction fragment and automated CE fraction collection can be used as a general procedure for sequencing unknown genomic DNA as well as mutated DNA mixtures.