DNA separation

Haab B B and Mathies R A 1999 Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams Ana/. Chem. 71 5137-45... 

Replication fork (Sechon 28 9) Point at which strands of double helical DNA separate... 

Boyd, BM Prausnitz, JM Blanch, HW, High-Frequency Altemating-Cross-Field Gel Electrophoresis with Neutral or Shghtly Charged Interpenetrating Networks to Improve DNA Separation, Electrophoresis 19, 3137, 1998. 

Auroux et al. give an up-to-date description of the application of pTAS components and systems, including cell culture and cell handling, immunoassays, DNA separation and analysis, polymerase chain reactions, and sequencing [43] (see also [44] for a description of the pTAS components and systems). 

In addition, the SRM contains a DNA molecular size standard for sizing the allele fragments a set of quantitative DNA standards in concentrations of 6-250 ng/6 pL and a visualization marker set which produces twelve bands ranging from 594 to 35 937 base pairs and which is used to assess the DNA separation on the electrophoretic gel. 

Hirabayashi, J. and Kasai, K.-I., Applied slalom chromatography. Improved DNA separations by the use of columns developed for reversed-phase chromatography, /. Chromatogr. A, 722, 135, 1996. 

Heller, C., Pakleza, C., and Viovy, J. L., DNA Separation with field inversion capillary electrophoresis, Electrophoresis, 16, 1423, 1995. 

Grossmann, P. D. and Soane, D. S., Experimental and theoretical studies of DNA separations by capillaryelectrophoresis in entangled polymer solutions, Biopolymers, 31, 1221, 1991. 

Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

Radioactive isotopes provide a very convenient way of monitoring the fate or metabolism of compounds that contain the isotopes. When used in this way, the isotope is described as a tracer and compounds into which the radioactive atom has been introduced are said to be labelled or tagged. The labelled molecules need only comprise a very small proportion of the total amount of the unlabelled radioactive substance because they act in the same way as the non-radioactive substance but can be detected very much more easily. The varied applications of tracers in biochemistry range from studies of metabolism in whole animals or isolated organs to sensitive quantitative analytical techniques, such as radioimmunoassay. Phosphorus-32 is used in work with nucleic acids, particularly in DNA sequencing and hybridization techniques. In these instances the isotope is used as a means of visualizing DNA separations by autoradiographic techniques. 

DNA separated into bands of different sized fragments... 

Figure 4. A rule that selects rotors to minimize the run time in a plasmid DNA separation. The rule examines a set of rotors called USERS.MATCHED.ROTORS, selecting those rotors that satisfy criteria based on the rotor design, tube volume, and k factor.

In CGE the capillary is filled with a gel containing cross-linked or linear polymers. The gel thereby acts as molecular sieve. Traditionally, cross-linked polyacrylamides and agarose have been utilized in the slab and tube format. Polyacrylamides when cross-linked have smaller pore sizes and are used for protein separations. The larger pore sizes of agarose gels are more suitable for DNA separation. Polyacrylamides yield very viscous gels. Therefore, polymerization is usually done on column, which has a lot of practical problems. 

DNA assays will certainly play an essential role in future medical research, along with proteomics. This makes high-throughput screening methods necessary. Parallel DNA separation chips coupled with high-sen-sitivity detection such as LIF or mass spectrometry (56) should be able to provide the required structural information in less time than with techniques currently employed (12). 

Slentz et al. [133] described the effects of geometry (size, shape, and dimensions) on the performance of COMOSS. Vreeland and Barron [134] described the design of functional materials for genomic and proteomic analyses in NCE. The authors discussed different polymer chemistries for micro-channel surface passivation and improved DNA separation. 

Smith et al. [27] described DNA separations in NCE with automated capillary sample introduction and laser-induced fluorescence (LIF) detection within... 

Tabuchi M, Baba Y (2005) Anal Chem 77 7090 Design for DNA separation medium using bacterial cellulose fibrils... 

The mechanism of separation with linear polymers is as follows. At a certain polymer concentration known as the entanglement threshold, the individual polymer strands begin to interact with each other, leading to a meshlike structure within the capillary. This allows DNA separation to take place. Many of the common polymers are cellulose derivatives, such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, and methylcellulose. Other applicable polymers include linear polyacrylamide, polyethylene oxide, agarose, polyvinyl pyrrolidone, and poly-N. Ar-dimethylacrylamide. High-resolution separation up to 12,000 bp has been reported using entangled polymer solutions. 

The aforementioned injection of a solution plug is termed as plug injection. Another sample introduction mode is stack injection. Stack and plug injections used in DNA separation have been compared (see Figure 4.2). Under similar conditions, the plug injection produced a better resolution, whereas the stack injection produced a higher sensitivity (see Chapter 6, section 6.2 for more on CE separation) [315]. 

Various sieving matrices for DNA fragment separation in a glass chip were evaluated. It was found the performance of HEC was comparable to PDMA, but was superior to polyacrylamide and PEO [611]. Hydroxylpropylcellulose (HPC), instead of HEC or HPMC, was used as a sieving matrix to achieve better DNA separations. In addition, the lower viscosity of HPC allows for more concentrated solution to be used without filling problems. CGE separation of DNA for analyzing mutations associated with Duchenne muscular dystrophy (DMD) was demonstrated [612],... 

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