Life Science 2001

April 2001 QuickComb-96

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 A TECHNICAL APPLICATION NEWSLETTER VOL 2   ISSUE 2    April 2001

 Molecular Biology Application Notes


QuickCombTM-96: A convenient Tool that Improves Sequence Data

By Jessica Copeland,
Sigma-Aldrich Corporation, St. Louis, MO USA

Introduction

Advances in sequencing technologies have expanded options for researchers with high, low or medium throughput. Although capillary electrophoresis may currently be the popular tool for high-throughput sequencing, slab-gel technology is still widely used. Whether it is slab-gel or capillary electrophoresis, the driving force to understand the genome will continue to fuel the development of technologies that allow researchers to optimize their work. Reviewing the gel-loading process has allowed us to develop a product that saves time and improves results.

The QuickComb™-96 is a porous membrane comb with laminated backing that allows sequencing samples to be conveniently loaded and stored on the same product. Using standard single or multichannel pipettors, sequencing reactions are loaded onto the teeth of the comb while on the lab bench. This eliminates the tedious step of accurately loading 96 samples with expensive syringe loaders. Once the samples are loaded, the QuickComb™-96 is simply inserted into the gel for electrophoresis. A loaded comb can be stored at 4 °C for up to 2 weeks or at —20 °C for up to 6 months without loss of read length or signal strength. The QuickComb™-96 provides sequencing laboratories with significant savings of time and money without sacrificing results.

In this report, we compare automated DNA sequence data obtained from samples loaded with QuickComb™-96 to those obtained from a conventional shark's-tooth comb. In addition, we outline the recommended procedure for using QuickComb™-96 for automated DNA sequencing.

Materials and Methods

All materials were supplied by Sigma-Aldrich Corporation (St. Louis, MO) unless otherwise stated.

The cycle sequencing reactions were prepared following the standard procedure using the ABI Prism® BigDye™ Terminator Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA). Because fewer cycles consistently yielded better results, the number of cycles was modified from the recommended 30 cycles to 25 cycles. The completed reactions were purified using the SigmaSpin™ columns.

Sequencing reactions were separated on a 4.5% AutoPAGE™ Plus gel prepared according to standard procedures with the exception that the casting comb was not secured. A less tight fit aids in inserting the QuickComb™-96 into the gel loading area. After gel polymerization and a successful plate check, 594 ml of deionized water was added to the upper buffer chamber. The lower buffer chamber was filled with 1X TBE as usual. The pre-run was initiated and immediately paused to allow the gel to reach 51 °C. The sequencing reactions were resuspended, denatured, and loaded on the QuickComb™-96 while the gel was warming to temperature. Once the gel reached the desired temperature (51 °C), the QuickComb™-96 was inserted and the desired run module (36E-1200) initiated. Current was applied for approximately 45 seconds to electro-elute the samples from the comb followed by comb removal, addition of 10X TBE (66 ml) to the upper buffer chamber, and electrophoresis resum ption. After the run was completed, the lanes were tracked and analyzed.

Results and Discussion

QuickComb™-96 vs. Shark's-tooth loading

When using a standard shark's-tooth comb to load samples, the researcher must insert the comb perfectly to ensure equal sample loading areas. If there is a small vertical difference from one end of the gel to the other, there is substantial variation in the quantity of sample that can be loaded in each lane. This results in the researcher's inability to equally compare all of the samples on a single gel. Another problem results from loading large numbers of samples. This requires that the first samples loaded must wait until the last sample is loaded to begin electrophoresis, allowing diffusion of the first samples to neighboring wells and into the upper buffer chamber. Lane-to-lane leakage is most serious when the shark's-tooth comb does not adequately seal between the wells. This can cause the bands to appear faint or sample contamination from adjacent lanes can make it nearly impossible to successfully analyze a lane. To combat this, combs must fit the gel space very snugly. This can cause comb i nsertion to be difficult easily leading to comb damage. It is not uncommon to find drawers full of damaged combs in sequencing facilities. QuickComb™-96 eliminates these pitfalls. With QuickComb™-96, all samples are simultaneously introduced to the gel. There is no concern for early vs. late diffusional processes leading to variable signal strengths. Additionally in the absence of an electric field, samples only slowly diffuse from the QuickComb™-96 resulting in highly defined lanes that are easier to track and analyze (Figure 1).

QuickComb™-96 Method

When using the QuickComb™-96, it is necessary to begin electrophoresis with only water in the upper buffer chamber. Operationally, after the comb has been inserted electrophoresis is commenced for 45 seconds followed by comb removal, buffer addition, and continued electrophoresis. As shown in (Figure 2), loading from 1X TBE causes the bands to be very broad and essentially un-assignable. Water loading produces very sharp easily assigned bands. This observation is most easily rationalized by the fact that the lower concentration of ions in water compared to 1X TBE results in the DNA having a higher mobility in water. In water, the samples on the QuickComb™-96 move more quickly from the pores of the comb resulting in sharper, more highly resolved sequence. The same mechanism could reasonably be the cause for the increase in read length f or QuickComb™-96 vs. traditional shark's-tooth loading (see Increased PHRED Scores).

Increased PHRED Scores

We observed that the QuickComb™-96 routinely yielded an additional 40-50 bases of usable sequencing data compared to traditional loading methods (Figure 3). To quantify this, we compared the QuickComb™-96 (cut in half) to standard shark's-tooth (also cut in half) loading on a single 4.5% AutoPAGE™ Plus gel. The gel was prepared for a water load followed by standard shark's-tooth loading after buffer addition to the upper buffer chamber. This experiment revealed a 12% increase in bases successfully analyzed with the QuickComb™-96. This is easily rationalized by the DNA fragments entering the gel from a lower dielectric medium resulting in more highly concentrated DNA at the outset. This concentrating effect would be expected to produce narrower band widths and thus better band resolution.

Conclusions

The QuickComb™-96 methodology continuously outperformed standard shark's-tooth loading. It provides gel uniformity, high signal strengths, and superior lane definition. Though developed with sequencing in mind, it can and should be used for loading any fluorescent DNA onto an automated sequencing machine. Applications such as genotyping and PCR product analysis have been successfully aided by using the QuickComb™-96.

Acknowledgements

The author would like to thank Dr. Brian Ward (Principal Scientist in Sequencing R&D), Tony Favello (Technology Transfer Manager), and Joel White (Technical Marketing) at Sigma-Aldrich Corporation, St. Louis, MO for their helpful discussions. Special thanks to Brandon Blakey for his collaborative efforts (Field Application Specialist, Applied Biosystems, St. Louis, MO).

About the Author

Jessica Copeland, B.S., is an associate scientist in Sequencing R&D at Sigma-Aldrich, St. Louis, MO.

ORDERING INFORMATION AND RELATED PRODUCTS

Product Code

Product Name

Unit

C3226

QuickComb™-96

10 each

 

 

50 each

     

P8977

AutoPAGE™ 4.5%

100 ml

 

 

5 x 100 ml

 

 

5000 ml

 

 

1 liter

     

T4415

Tris-Borate-EDTA Buffer 10X Concentrate (TBE)

4 liter

 

 

10 liter

Life Science Catalog 2000-2001, pages 119 (P8977), 123 (T4415), 125-127 (Glass Plates) and 128 (C3226).


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