Electrophoresis

571

Chapter Outline

23.1 Introduction: The Human Genome Project

23.1A What Is Electrophoresis?

23.1B How Is Electrophoresis Performed?

23.2 General Principles of Electrophoresis

23.2A Factors Affecting Analyte Migration

23.2B Factors Affecting Band-Broadening

23.3 Gel Electrophoresis

23.3A What Is Gel Electrophoresis?

23.3B How Is Gel Electrophoresis Performed?

23.3C What Are Some Special Types of Gel Electrophoresis?

23.4 Capillary Electrophoresis

23.4A What Is Capillary Electrophoresis?

23.4B How Is Capillary Electrophoresis Performed?

23.4C What Are Some Special Types of Capillary Electrophoresis?

Chapter 23

Electrophoresis

23.1 INTRODUCTION: THE HUMAN

GENOME PROJECT

February 2001 saw one of the greatest achievements of

modern science. It was at this time that two scientific

papers appeared, one in the journal Science and the other

in Nature, reporting the sequence of human DNA (or the

“human genome”).1,2 These papers were the result of a

major research effort known as the Human Genome

Project, which was formally begun in 1990 under the

sponsorship of the U.S. Department of Energy and the

National Institutes of Health.3

Although it was anticipated to take 15 years to fin￾ish, this project was “completed” in about a decade. This

early completion was made possible by several advances

that occurred in techniques for sequencing DNA. One

common approach for sequencing DNA is the Sanger

method (see Figure 23.1). In the Sanger method, the sec￾tion of DNA to be examined (known as the “template”) is

mixed with a segment of DNA that binds to part of this

sequence (the “primer”). This mixture is placed into four

containers that have the nucleotides and enzymes

needed to build on the template. These containers also

have special labeled nucleotides that will stop the elonga￾tion of DNA after the addition of a C, G, A, or T to its

sequence. The DNA strands formed in each container are

later separated according to their size. By comparing the

length of these strands and by knowing which labeled

nucleotides are at the end of each strand, the sequence of

the DNA can be determined.4

The Sanger method was originally developed as a

manual technique that took long periods of time to per￾form. Thus, one thing that had to be addressed early in

the Human Genome Project was the creation of faster,

automated systems for sequencing DNA.5,6 Both tradi￾tional and newer systems for accomplishing this

sequencing utilize a separation method known as

electrophoresis. In this chapter we learn about elec￾trophoresis, look at its applications, and see how

improvements in this technique made the Human

Genome Project possible.

23.1A What Is Electrophoresis?

Electrophoresis is a technique in which solutes are sepa￾rated by their different rates of migration in an electric

field (see Figure 23.2).7–10 To carry out this method, a

sample is first placed in a container or support that also

contains a background electrolyte (or “running buffer”).

When an electric field is later applied to this system, the

ions in the running buffer will flow from one electrode to

the other and provide the current needed to maintain the

applied voltage. At the same time, positively charged

ions in the sample will move toward the negative elec￾trode (the cathode), while negatively charged ions will

move toward the positive electrode (the anode). The

result is a separation of these ions based on their charge

and size. Because many biological compounds have

charges or ionizable groups (e.g., DNA and proteins),

electrophoresis is frequently utilized in biochemical and

96943_23_ch23_p571-596 1/8/10 2:54 PM Page 571

572 Chapter 23 • Electrophoresis

Primer

Sample of DNA

Add DNA and primer to

four reaction mixtures

for replication,

each mixture containing

a different elongation￾stopping nucleotide

Stops

at C

Stops

at T

Stops

at A

Stops

at G

Mixtures of elongated primer strands with

various lengths and stopped at different

nucleotides

Sequence of

original DNA

Separate primers strands by

size

usin

g electrophoresis

Stopped

at C

Stopped

at T

Stopped

at A

Stopped

at G

G

T

G

A

C

T

A

G

T

C

G

A

T

(a)

(b)

DNA replication

Separate and analyze primer strands

FIGURE 23.1 Sequencing of DNA by the Sanger method. This method is named after F. Sanger, one of the

scientists who originally reported this technique.4 The final DNA sequence is determined in this method by

looking at the sequence of the primer strands and using the complementary nucleotides (C for G, A for T, G

for C, and T for A) to describe the sequence of the original DNA.

of moving boundaries between regions that contained dif￾ferent mixtures of proteins, as shown in Figure 23.3.10,16

Today it is more common to use small samples to allow

analytes to be separated into narrow bands or zones, giving

a method known as zone electrophoresis.

8–10,16 An example of

zone electrophoresis is shown in Figure 23.1, where DNA is

sequenced by separating its strands of various lengths into

narrow bands on a gel.

There are many ways in which electrophoresis is

used for chemical analysis. These include the sequencing

of DNA, as well as the purification of proteins, peptides,

and other biomolecules. In clinical chemistry, elec￾trophoresis is an important tool for examining the pat￾terns of amino acids, serum proteins, enzymes, and

lipoproteins in the body. Electrophoresis is also used in

the analysis of organic and inorganic ions in foods, com￾mercial products, and environmental samples. In addi￾tion, electrophoresis is an essential component of medical

and pharmaceutical research for the characterization of

medical research. This approach can also be adapted for

work with small ions (like or ) or for large

charged particles (such as cells and viruses).

Even though it has been known for one hundred years

that substances like proteins and enzymes have a character￾istic rate of travel in an electric field,11–13 electrophoresis did

not become a routine separation method until around the

1930s. One notable advance occurred in 1937 when a scien￾tist named Arne Tiselius (Figure 23.3) used electrophoresis

for the separation of serum proteins.3,14 Tiselius conducted

this separation by employing a U-shaped tube in which he

placed his sample and running buffer. When he applied an

electric field, proteins in the sample began to separate as

they migrated toward the electrodes of opposite charge.

However, the use of a large sample volume gave a series of

broad and only partially resolved regions that contained

different mixtures of the original proteins.15

The method employed by Tiselius is now known as

moving boundary electrophoresis, because it produced a series

NO3

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