Is Higher Concentration of Linear Polyacrylamide Not Suitable for the Capillary Electrophoretic Separation of Large DNA?

Abstract

Polyacrylamide is an excellent separation matrix for capillary electrophoresis 110 and conventional gel electrophoresis,11,12 Low crosslinked polyacrylamide gel and linear polyacrylamide gave an excellent resolution of double-stranded DNA fragments using capillary electrophoresis. l-4,9,10 Linear polyacrylamide has been used as a separation matrix for DNA sequencing especially for long reading by capillary electrophoresis.5-gIn our common knowledge, higher concentration of polyacrylamide is suitable for separating small DNA fragments and lower concentration of polyacrylamide is indispensable for the separation of large DNA fragments.11-13 Low concentration of polyacrylamide gel (3% T and 0.5% C) is applied to the separation of up to 12 kbp DNA fragments1,2,4 and 1.5% linear polyacrylamide gave a fine resolution of DNA fragments up to 23 kbp3, whereas shorter DNA fragments up to 1 kbp should be separated by using 6% linear polyacrylamide.l,3 Large DNA fragments up to 48.5 kbp could only be separated by using very diluted linear polyacrylamide (0.6%).9 Use of lower concentration of linear polyacrylamide (2%) allowed DNA sequencing of more than 1000 bases per rung, whereas DNA sequencing of less than 500 bases was achieved using 6% linear polyacrylamide as a separation matrix.5,6More recently, some cellulose derivatives were proven to be an efficient separation matrix for DNA fragments.l3-19 In this instance, use of ultradilute polymer solution (0.00125%) was necessary for the separation of DNA up to 23 kbp17-19, whereas 0.5% cellulose solution is suitable for the separation of DNA fragments up to 1 kbp.14-16 A pulsed field technique20 coupled with use of ultradilute polymer solution gave an excellent separation of multikilobase and mega base DNA fragment2l-26, which could not be separated without a pulsed filed technique.23Both standard textbooks on gel electrophoresis11,12 and comprehensive review on capillary electrophoresisl3 recommend that effective range of separation of DNA fragments is less than 300 -400 by using 8% polyacrylamide in both slab gel electrophoresis and capillary electrophoresis. Additionally, these textbooks and review conclude the use of less than 3% polyacrylamide is essential for the separation of large DNA more than 1 kbp.In this paper, we demonstrate that 7% polyacrylamide is suitable for the rapid separation of large DNA fragments up to 50 kbp by capillary electrophoresis without pulsed electric field. Only 5.4 min is required for the complete resolution of seven DNA fragments in a sample of DNA restriction fragments T4dC/ Bgl I digest containing DNA fragments ranging from 1.26 to 49.31 kbp by constant field capillary electrophoresis and the peak of T4dC DNA (166 kbp) appears at ca. 8.3 min. Such large DNA fragments up to 50 kbp have never been separated without use of lower concentration of separation matrix and pulsed field technique.21-26 Additionally, DNA conformational dynamics in high concentration of polyacrylamide under electric field was observed directly by use of fluorescence...

Electrophoretic mobility of linear and star‐branched DNA in semidilute polymer solutions

“…For linear DNA we found that for all LPA M w investigated the linear DNA conformation changed from dominant U-to I-shape as LPA concentration is increased as reported earlier (see Fig. 8c-d) [40,43]. We have also found that the concentration around which the transition from U-to I-shape conformation is induced decreases with increasing LPA M w (see Fig.…”

“…Furthermore, the transition from U-dominant to I-dominant conformations observed (around E < 15 V/ cm for all 2% w/v LPA solutions) produces no noticeable change in the field-dependence of the mobility. Ueda and Baba [40] reported a similar observation, namely that the electrophoretic mobility of linear DNA changes little when it undergoes a transition from U-to I-dominant conformations during electrophoresis. These results are evidently similar to our previous observation that conformation differences between electrophoresing star-branched and linear DNA have at most a negligible effect on mobility in unentangled LPA solutions.…”

“…A series of videomicroscopy studies [17,[40][41][42][43][44][45] of large linear DNA in polymer solutions has revealed that the DNA molecule undergoes significant conformational changes during electrophoretic migration. Carlsson et al [43] and Morris and co-workers [44,45] in their visualization studies found that, although the dynamics of DNA migrating through polymer solutions have some characteristics similar to that of DNA migrating in gels, the apex of the U-shape conformation for linear DNA migrates in polymer solution whereas in gels the apex remains stationary.…”

“…Moreover, short matrix polymers must be present at concentrations higher than long ones to induce similar DNA conformational changes, indicating that the parameter determining DNA conformation is not the absolute polymer concentration but the degree of polymer entanglement [43]. Ueda and Baba [40] argued that the formation of U-shape conformation in semidilute polymer solution is due to the short lifetime of interactions between migrating DNA and matrix polymer chains hooked near the end of the DNA, in comparison to matrix polymer chains near the mid-section of the migrating DNA molecule, where the apex of the U is formed. From observations of changes in the DNA segment density along the backbone during Ishaped migratory motion as a function of time, these authors concluded that the LPA chains remain at essentially fixed positions despite the fact that they are being perpetually pushed by DNA segments in concentrated (,10c*) LPA solution.…”

Sieving mechanisms in polymeric matrices

“…x b , the presence of reptation-type migration in highly entangled solutions was strongly supported by a series of videomicroscopy studies of the motion of large DNA molecules [113][114][115][116].…”