mass is important in developing new sieving matrices. Polysaccharides such as hydroxyethyl cellulose can be analyzed by using SEC-MALLS. Linear polymers (Mr 107and more) elute in the void peak in SEC and have to be measured by micro batch MALLS.
 

 

DNA sequencing by CE

Capillary electrophoresis in sieving polymer solutions is a tool for separation of DNA sequencing fragments.  DNA molecules terminated at the same base are labelled with the same fluorescent dye. Samples are  separated in a capillary electrophoresis instrument. Electropherogram of DNA sequencing fragments consists of 4 independent tracks for each base. When put together it can be used to determine the sequence of bases in DNA (Fig. 1).

 

 

Figure 1.  Four color electropherogram of DNA sequencing fragments.

 

 

Selection of sieving polymer

Sieving polymers for DNA sequencing have been selected more or less empirically. Linear polyacrylamide (Ruiz-Martinez et al., 1993), polyethylene oxide (Fung and Yeung, 1995), hydroxyethyl cellulose (Bashkin et al., 1996),  polydimethyl acrylamide (Madabhushi, 1998), polyvinyl pyrrolidone (Gao et al., 1999) are those used most frequently. Although pore size in polymer solution depends on the polymer concentration according to a theory (Grossman and Soane, 1991), the sieving performance actually depends on the molecular mass of the polymer, too. This is because of constraint release (Viovy et al., 1993)
 

and network dissociation by forces caused by migrating DNA fiber (Bae and Soane, 1991).

 

 

 

Molecular mass determination

Molecular mass of sieving polymers are determined by size-exclusion chromatography (SEC)- multi angle laser light scattering (MALLS) (Fig. 2).

 

For high molecular mass polymers, dn/dc has to be determined; only 76% recovery of LPA was observed.

 

Figure 2. SEC-MALLS setup for determination of molecular mass of sieving polymers.

 

Table 1. Dn/dc of measured polymers (30°C)

 

linear polyacrylamide                     0.153

epoxypolydimethylacrylamide       0.147

guaran                                               0.189

 

To avoid shearing at SEC, the flow rate has to be reduced to 0.2 mL/min. The Berry formalism with second degree fit was found to be optimum for the determination of high molecular masses (Fig. 3).

 

Figure 3. Debye plot for linear polyacrylamide.

 

If the molecular mass of polymer such as LPA exceeds 107, SEC-MALLS gives falsely lower results and microbatch mode has to be used (Barron et al. 2000). Whereas SEC-MALLS provides the weight molecular mass of a LPA preparation of about 107, the micro batch mode gives the weight molecular mass over 2 x 107 (Fig. 4).

Figure 4. Zimm  plot for linear polyacrylamide.

 

Quest for an ideal polymer

Selectivity as a real engine for separation in  electrophoresis, is related to the slope of log-log mobility curve of DNA (Fig. 5). The slope of the log-log mobility curve at its inflection point can be used to express sieving properties of a polymer quantitatively (Dolnik & Gurske 1999).

 

Figure 5. Log log mobility curves of DNA in HEC, Mr  1.3 x 106.

 

Double reciprocal plot

A double reciprocal plot of the inflection slope and polymer concentration provides KD values that express the sieving properties of a polymer independently of its concentration. (Fig. 6). KD is in fact a concentration of polymer, at which the inflection slope equals to half of the inflection slope at the infinite concentration of polymer.

 

Figure 6. Double reciprocal plot of inflection slope and concentration of hydroxyethyl cellulose.

 

 

 

KD and intrinsic viscosity

KD has been found to be reciprocally related to intrinsic viscosity of sieving polymer as determined by viscosimetry (Fig. 7). Sieving properties of a polymer can be predicted from the value of the intrinsic viscosity obtained for a given polymer. However, we could not find a good correlation of data for intrinsic viscosity obtained by MALLS and viscosimetry.

 

Figure 7. Relationship between KD and intrinsic viscosity for HEC.

 

 

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Abstract

Capillary electrophoresis in replaceable polymer solutions has become a standard method for the sequencing of DNA. The performance of the sieving polymers is strongly related to their molecular mass and determination of molecular
 

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