Capillary Electrophoresis of Nucleic Acids [Vol 2] [Methods in Molec Bio 163] - K. Mitchelson, J. Cheng (Humana, 2001) WW.pdf - książki - Bio Med 05 - bibiariel

Methods in Molecular Biology TM

VOLUME 163

Capillary

Electrophoresis

of Nucleic Acids

Volume II

Practical Applications

of Capillary Electrophoresis

Capillary

Electrophoresis

of Nucleic Acids

Volume II

Practical Applications

of Capillary Electrophoresis

Edited by

Keith R. Mitchelson

Jing Cheng

Edited by

Keith R. Mitchelson

Jing Cheng

HUMANA PRESS

Rapid DNA Fragment Analysis by CE

Development of a High-Throughput Capillary

Electrophoresis Protocol for DNA Fragment Analysis

H. Michael Wenz, David Dailey, and Martin D. Johnson

1. Introduction

Since the first descriptions of electrophoresis in small diameter tubes in the 1970s and

1980s (1 , 2) , capillary electrophoresis (CE) has been recognized for its potential to replace

slab-gel electrophoresis for the analysis of nucleic acids (3 , 4) . In particular, the availability

of commercial instrumentation for CE over the last several years has made both the size

determination and quantitation of DNA restriction fragments or polymerase chain reaction

(PCR) products amenable to automation. Due to the same charge-to-mass ratio, the elec-

trophoretic mobility of nucleic acid molecules in free solution is largely independent of

their molecular size (5) . Therefore, a sieving medium is required for the electrophoretic

analysis of DNA fragments based on their size. Typically, two different principal types of

separation matrix are used. The first type of matrix is of high viscosity polymer (e.g.,

polyacrylamide) with a well-defined crosslinked gel in regard to the structure and size of

its pores. The second type of matrix is a noncrosslinked linear polymer network of materi-

als such as, linear polyacrylamide, agarose, cellulose, dextran, poly(ethylene oxide), with

lower viscosity than the former type and with a more dynamic pore structure. Although the

first type of matrix is attached covalently to the capillary wall and may provide better

separation for small (sequencing) fragments, the second matrix format has the advantage

of being able to be replenished after each electrophoretic cycle. This typically extends the

lifetime of a capillary, prevents contamination of the system, avoids sample carryover and

allows the use of temperatures well above room temperature. Most matrices used in both

systems are tolerant to the addition of DNA denaturants. Many different media useful for

the separation of DNA have now become commercially available (6) .

In summary, the application of CE for DNA related research is attractive for

numerous reasons:

The high degree of automation avoids cumbersome gel pouring and sample loading.

High mass sensitivity eliminates the need to label DNA with carcinogenic stains, or with

radioactive DNA precursors.

From: Methods in Molecular Biology, Vol. 163:

Capillary Electrophoresis of Nucleic Acids, Vol. 2: Practical Applications of Capillary Electrophoresis

Edited by: K. R. Mitchelson and J. Cheng © Humana Press Inc., Totowa, NJ

Wenz, Dailey, and Johnson

3. Very reproducible size information is achieved through the use of an internal size stan-

dard, which compensates for run-to-run variations.

4. Quantitative information is obtained after on-line detection.

5. Differences in fragment length as small as one base can be visualized by utilizing appro-

priate separation conditions.

1.1. Fast-Cycle CE

Typical DNA separations by CE are considered fast, ranging from 10 to 60 min.

However, single capillary instruments do not achieve the same productivity as slab

gels, which have longer run times, but have higher throughput owing to a multitude of

simultaneously addressable lanes. Attempts have been made to substantially decrease

the run times in capillaries by using very short effective lengths and high electric field

strength (7 , 8 , 9) . These approaches considerably shorten the electrophoresis times to

3 min or less. However, none of these protocols has been implemented on a commer-

cially available instrument.

We have developed a “fast-cycle capillary electrophoresis protocol” to address the

need for high throughput and to make it amenable for commercially available instru-

mentation. This protocol allows the electrophoretic separation of DNA fragments up

to 500 bp in length in less than 5 min with a total cycle time from one sample injection

to the next of approx 7 min.

Analyses are performed on the ABI PRISM ® 310 Genetic Analyzer that allows the

simultaneous analysis of fragments that are tagged with different fluorophors. In order

to achieve fast analysis times, several conventional electrophoresis factors are

modified:

Both the separation polymer (2%) and electrophoresis buffer (60 m M ) are at low concen-

tration to accelerate electro-migration.

Electrophoresis run temperature is elevated to 60°C, with DNA molecules separated as

single-strands.

The capillary length is shortened (effective separation length of 30 cm).

We show that typically 306 consecutive injections can be performed under these

conditions without the need to change either the capillary or the electrophoresis buffer.

This protocol can be used for applications that require the resolution of fragments that

differ by at least 5 bp in length with a sizing precision of 0.4 bp. We present data that

demonstrate the use of this protocol for the sizing and quantification of PCR frag-

ments, the analysis of minisequencing reactions, the analysis of DNA fragments that

are the product of an oligonucleotide ligation assay (OLA), and the quality control of

phosphorylated short synthetic oligonucleotides.

2. Materials

2.1. Instrumentation and Electrophoresis

The ABI PRISM ® 310 Genetic Analyzer (PE Biosystems, Foster City, CA), a laser-based

CE instrument, is used for all experiments. This instrument uses a multi-line argon-ion

laser, adjustable to 10 mW, which excites multiple fluorophores at 488 and 514 nm.

Fluorescence emission is recorded between 525 and 650 nm on a cooled CCD camera.

This configuration currently allows the multiplexing and sizing of samples that overlap in

Rapid DNA Fragment Analysis by CE

size by using three different fluorophors, plus an additional fluorophore that is attached to

an internal size standard.

3. The instrument controls temperature between ambient and 60°C with an accuracy of

± 1°C.

4. Electrophoresis voltage is controlled between 100 and 15,000 V.

5. A sample tray holds 48 or 96 samples for unattended operation.

6. Data are collected and automatically analyzed, using an instrument specific collection

software and GeneScan analysis software (PE Biosystems, Foster City, CA).

7. The separation medium in the capillary is automatically replaced after each sample run.

Samples are introduced by electrokinetic injection, typically for 5–10 s at 7–15 kV.

8. The features that allow the use of this high throughput protocol are implemented in the

PRISM 310 ® Collection Software, version 1.2 ( see Note 1 ).

3. Methods

3.1. Polymer Preparation

GeneScan polymer (PE Biosystems, Foster City, CA) is a hydrophilic polymer that pro-

vides molecular sieving and noncovalent wall coating, when used in uncoated fused silica

capillaries (PE Biosystems, Foster City, CA) ( see Note 2 ).

GeneScan polymer is provided as a 7% stock solution in water that can be diluted and

mixed with different additives, such as urea or glycerol. The polymer is most commonly

diluted in Genetic Analyzer Buffer containing EDTA (PE Biosystems, Foster City, CA),

but is also compatible with other buffers (10) .

To prepare a 2% solution of GeneScan polymer, combine 14.3 mL of the polymer and

3 mL Genetic Analyzer buffer with EDTA in a 50-mL polypropylene tube, bring to

50 mL with deionized water and mix thoroughly. For the preparation of the 0.6X electro-

phoresis buffer, combine 3 mL of the Genetic Analyzer buffer with EDTA with 47 mL of

distilled water. Both solutions are stable for at least 4 wk refrigerated at 4

C. Before use,

the solutions have to be warmed up to room temperature.

3.2. Sample Preparations

3.2.1. PCR Samples

To evaluate the robustness of the fast protocol, five short tandem repeat (STR) markers

with repeat units of 4 bp are individually amplified by PCR. Samples are labeled with 6-Fam

(blue), Hex (green), and Ned (yellow). Markers are pooled in a ratio to provide compa-

rable intensities when injected into the capillary.

Four µL of the pool are added to 15 µL of deionized formamide and 0.25 µL of GeneScan

500 size standard, labeled with Rox (red). Up to 16 injections are performed from each

sample tube. Samples are injected for 30 s at 15 kV.

It is critical to dilute the oligonucleotide sample into high-quality deionized formamide

for loading onto the CE instrument. To deionize formamide, mix 50 mL of formamide

and 5 g of AG501 X8 mixed bed resin and stir for at least 30 min at room temperature.

Check if the pH of formamide is greater than 7.0. If it is not, repeat above step. When the

pH is greater than 7.0, dispense the deionized formamide into aliquots of 500

L and

store for up to 3 mo at –15 to –25

C. Usually, there is no need to purify the DNA sample

before diluting it into deionized formamide. Should a signal, even with extended injec-

tion time/voltage prove to be insufficient, purifying the sample, and thereby removing

salt anions that might compete with the DNA sample during electrokinetic injection, might

increase the DNA signal.

Wenz, Dailey, and Johnson

3.2.2. Minisequencing Samples

1. Minisequencing reactions are generated in a single tube using 5 µL of the SNaPshot

minisequencing reaction premix (PE Biosystems, Foster City, CA) along with 0.15 pmol

of primer for the A, C, and T reaction and 0.75 pmol primer for the G reaction. pGEM

(0.4 µg) is used as template.

2. Following primer extension, reactions are treated with 0.5 U shrimp alkaline phosphatase

(SAP) (USB, Cleveland, OH) to modify the mobility of the unincorporated fluorescently

labeled ddNTPs.

3. One µL of the SAP treated sample is diluted into 10 µL of deionized formamide. Samples

are injected for 5 s at 15 kV.

3.2.3. Oligonucleotide Ligation Assay (OLA)

For the OLA reaction, a DNA sample heterozygous for locus 621+1 G/T of the CFTR

gene is interrogated with two allele specific probes and one common probe (11) .

The allele specific oligo (ASO) detecting wild-type is 17 nt long and labeled with 6-Fam,

the ASO detecting the mutation is 18 nt long and labeled with Vic (green); the common

probe is 41 nt long (including a 24-nt modifier sequence).

OLA conditions are essentially as described in ref. 11 , with the exception that 80 OLA

cycles are used. Typically, 0.5

L of the sample is diluted into 9

L of deionized

formamide. Samples are injected for 5 s at 15 kV.

3.2.4. Oligonucleotide Probes

Seven-mer oligonucleotides are synthesized in 50-nmol scale on a DNA synthesizer

Model 3984 (PE Biosystems, Foster City, CA) using standard amidite chemistry. Oligo-

nucleotides are labeled on the 3'-end with 6-Fam, followed by two random mixed base

sequences. The terminal 5'-nt is chemically phosphorylated through PhosphoLink reac-

tion (PE Biosystems, Foster City, CA). Unpurified oligonucleotides are analyzed by ion-

exchange high performance liquid chromatography (HPLC) and oligonucleotides with

less than 70% purity are discarded. Typically, 1

L of a sample is diluted into 9

L of

deionized formamide, and is then injected for 5 s at 15 kV.

3.3. Protocol Optimization

Our goal was to develop a CE protocol that provides at least 5-bp resolution between

DNA fragments as well as a fast-analysis time to detect DNA fragments in the size range

between 75 and 500 bp, the typical size range for PCR products. This protocol is useful to

confirm the presence or absence of an expected amplification product, provide informa-

tion about the quality of the amplification, and if necessary, allow the determination of

the ratio in peak height or area of adjacent DNA peaks ( see Note 3 ).

We started with a protocol that was previously recommended for the analysis of dsDNA

in the size range between 50 and approx 5000 bp under nondenaturing electrophoresis

conditions (12) . This protocol uses a hydrophilic polymer (GeneScan polymer) of low

viscosity, that accomplishes both the separation of DNA fragments and the dynamic coat-

ing of the capillary walls when used together with uncoated fused silica glass capillaries

( see Table 1 , #1). To monitor the effect of the described changes from the initial proto-

col, we injected a DNA ladder (GeneScan 500-size standard) into the capillary. This lad-

der consists of DNA fragments ranging in size from 50 to 500 bp; one of the strands is

labeled with the fluorophore Tamra (red). We determined the electrophoresis time for the

100-, 300-, and 500-bp fragments and calculated the resolution in the 150- and 500-bp

Capillary Electrophoresis of Nucleic Acids [Vol 2] [Methods in Molec Bio 163] - K. Mitchelson, J. Cheng (Humana, 2001) WW.pdf

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