Key points:

Polymerase chain reaction, or PCR, is a technique to make many

copies of a specific DNA region in vitro (in a test tube rather than an
organism).

 PCR relies on a thermostable DNA polymerase, Taq polymerase,

and requires DNA primers designed specifically for the DNA
region of interest.

 In PCR, the reaction is repeatedly cycled through a series of

temperature changes, which allow many copies of the target region
to be produced.

 PCR has many research and practical applications. It is routinely

used in DNA cloning, medical diagnostics, and forensic analysis of
DNA.

What is PCR?
Polymerase chain reaction (PCR) is a common laboratory
technique used to make many copies (millions or billions!) of a
particular region of DNA. This DNA region can be anything the
experimenter is interested in. For example, it might be a gene whose
function a researcher wants to understand, or a genetic marker used
by forensic scientists to match crime scene DNA with suspects.

Typically, the goal of PCR is to make enough of the target DNA

region that it can be analyzed or used in some other way. For
instance, DNA amplified by PCR may be sent for sequencing,
visualized by gel electrophoresis, or cloned into a plasmid for further
experiments.
PCR is used in many areas of biology and medicine, including
molecular biology research, medical diagnostics, and even some
branches of ecology.

Taq polymerase
Like DNA replication in an organism, PCR requires a DNA
polymerase enzyme that makes new strands of DNA, using existing
strands as templates. The DNA polymerase typically used in PCR is
called Taq polymerase, after the heat-tolerant bacterium from which
it was isolated (Thermus aquaticus).

T. aquaticus lives in hot springs and hydrothermal vents. Its DNA

polymerase is very heat-stable and is most active around 70 °\text
C70°C70, °, start text, C, end text (a temperature at which a human
or E. coli DNA polymerase would be nonfunctional). This heat-
stability makes Taq polymerase ideal for PCR. As we'll see, high
temperature is used repeatedly in PCR to denature the template
DNA, or separate its strands.

PCR primers
Like other DNA polymerases, Taq polymerase can only make DNA
if it's given a primer, a short sequence of nucleotides that provides a
starting point for DNA synthesis. In a PCR reaction, the
experimenter determines the region of DNA that will be copied, or
amplified, by the primers she or he chooses.

PCR primers are short pieces of single-stranded DNA, usually

around 202020 nucleotides in length. Two primers are used in each
PCR reaction, and they are designed so that they flank the target
region (region that should be copied). That is, they are given
sequences that will make them bind to opposite strands of the
template DNA, just at the edges of the region to be copied. The
primers bind to the template by complementary base pairing.

Template DNA:

5' TATCAGATCCATGGAGT...GAGTACTAGTCCTATGAGT 3'

3' ATAGTCTAGGTACCTCA...CTCATGATCAGGATACTCA 5'

Primer 1: 5' CAGATCCATGG 3' Primer 2:

When the primers are bound to the template, they can be extended
by the polymerase, and the region that lies between them will get
copied.
 The steps of PCR
The key ingredients of a PCR reaction are Taq polymerase, primers,
template DNA, and nucleotides (DNA building blocks). The
ingredients are assembled in a tube, along with cofactors needed by
the enzyme, and are put through repeated cycles of heating and
cooling that allow DNA to be synthesized.

The basic steps are:

1. Denaturation (96 °\text C96°C96, °, start text, C, end text): Heat the
reaction strongly to separate, or denature, the DNA strands. This
provides single-stranded template for the next step.

2. Annealing (555555 - 656565°\text C°C°, start text, C, end text):

Cool the reaction so the primers can bind to their complementary
sequences on the single-stranded template DNA.

3. Extension (72 °\text C72°C72, °, start text, C, end text): Raise the
reaction temperatures so Taq polymerase extends the primers,
synthesizing new strands of DNA.
This cycle repeats 252525 - 353535 times in a typical PCR reaction,
which generally takes 222 - 444 hours, depending on the length of
the DNA region being copied. If the reaction is efficient (works
well), the target region can go from just one or a few copies to
billions.

That’s because it’s not just the original DNA that’s used as a
template each time. Instead, the new DNA that’s made in one round
can serve as a template in the next round of DNA synthesis. There
are many copies of the primers and many molecules
of Taq polymerase floating around in the reaction, so the number of
DNA molecules can roughly double in each round of cycling. This
pattern of exponential growth is shown in the image below.
Using gel electrophoresis to visualize the
results of PCR
The results of a PCR reaction are usually visualized (made visible)
using gel electrophoresis. Gel electrophoresis is a technique in
which fragments of DNA are pulled through a gel matrix by an
electric current, and it separates DNA fragments according to size. A
standard, or DNA ladder, is typically included so that the size of the
fragments in the PCR sample can be determined.

DNA fragments of the same length form a "band" on the gel, which
can be seen by eye if the gel is stained with a DNA-binding dye. For
example, a PCR reaction producing a 400400400 base pair (bp)
fragment would look like this on a gel:
 Left lane: DNA ladder with 100, 200, 300, 400, 500 bp bands.

Right lane: result of PCR reaction, a band at 400 bp.

A DNA band contains many, many copies of the target DNA region,
not just one or a few copies. Because DNA is microscopic, lots of
copies of it must be present before we can see it by eye. This is a big
part of why PCR is an important tool: it produces enough copies of a
DNA sequence that we can see or manipulate that region of DNA.

Applications of PCR
Using PCR, a DNA sequence can be amplified millions or billions of
times, producing enough DNA copies to be analyzed using other
techniques. For instance, the DNA may be visualized by gel
electrophoresis, sent for sequencing, or digested with restriction
enzymes and cloned into a plasmid.

PCR is used in many research labs, and it also has practical

applications in forensics, genetic testing, and diagnostics. For
instance, PCR is used to amplify genes associated with genetic
disorders from the DNA of patients (or from fetal DNA, in the case
of prenatal testing). PCR can also be used to test for a bacterium or
DNA virus in a patient's body: if the pathogen is present, it may be
possible to amplify regions of its DNA from a blood or tissue
sample.

Sample problem: PCR in forensics

Suppose that you are working in a forensics lab. You have just
received a DNA sample from a hair left at a crime scene, along with
DNA samples from three possible suspects. Your job is to examine a
particular genetic marker and see whether any of the three suspects
matches the hair DNA for this marker.

The marker comes in two alleles, or versions. One contains a single

repeat (brown region below), while the other contains two copies of
the repeat. In a PCR reaction with primers that flank the repeat
region, the first allele produces a 200200200 \text{bp}bpstart text, b,
p, end text DNA fragment, while the second produces
a 300300300 \text{bp}bpstart text, b, p, end text DNA fragment:

Marker allele 1: primers flanking repeat region amplify a 200 bp

fragment of DNA

Marker allele 2: primers flanking repeat region amplify a 300 bp

fragment of DNA

You perform PCR on the four DNA samples and visualize the
results by gel electrophoresis, as shown below:
 The gel has five lanes:

First lane: DNA ladder with 100, 200, 300, 400, and 500 bp bands.

Second lane: DNA from crime scene, 200 bp band.

Third lane: Suspect #1 DNA, 300 bp band.

Fourth lane: Suspect #2 DNA, 200 and 300 bp bands.

Fifth lane: Suspect #3 DNA, 200 bp band.

Which suspect's DNA matches the DNA from the crime scene at
this marker?
 Key points:
 Gel electrophoresis is a technique used to separate DNA fragments
according to their size.

 DNA samples are loaded into wells (indentations) at one end of a

gel, and an electric current is applied to pull them through the gel.

 DNA fragments are negatively charged, so they move towards the

positive electrode. Because all DNA fragments have the same
amount of charge per mass, small fragments move through the gel
faster than large ones.

 When a gel is stained with a DNA-binding dye, the DNA fragments

can be seen as bands, each representing a group of same-sized DNA
fragments.

Introduction
Suppose you have just done a PCR reaction, making many copies of
a target DNA region. Or perhaps you’ve done some DNA cloning,
trying to "paste" a gene into a circular DNA plasmid.

Now, you want to check and see whether your PCR worked, or
whether your plasmid has the right gene in it. What technique can
you use to visualize (directly observe) the fragments of DNA?

Gel electrophoresis
Gel electrophoresis is a technique used to separate DNA fragments
(or other macromolecules, such as RNA and proteins) based on their
size and charge. Electrophoresis involves running a current through
a gel containing the molecules of interest. Based on their size and
charge, the molecules will travel through the gel in different
directions or at different speeds, allowing them to be separated from
one another.

All DNA molecules have the same amount of charge per mass.
Because of this, gel electrophoresis of DNA fragments separates
them based on size only. Using electrophoresis, we can see how
many different DNA fragments are present in a sample and how
large they are relative to one another. We can also determine the
absolute size of a piece of DNA by examining it next to a standard
"yardstick" made up of DNA fragments of known sizes.

What is a gel?
As the name suggests, gel electrophoresis involves a gel: a slab of
Jello-like material. Gels for DNA separation are often made out of a
polysaccharide called agarose, which comes as dry, powdered
flakes. When the agarose is heated in a buffer (water with some salts
in it) and allowed to cool, it will form a solid, slightly squishy gel.
At the molecular level, the gel is a matrix of agarose molecules that
are held together by hydrogen bonds and form tiny pores.

At one end, the gel has pocket-like indentations called wells, which
are where the DNA samples will be placed:
Before the DNA samples are added, the gel must be placed in a gel
box. One end of the box is hooked to a positive electrode, while the
other end is hooked to a negative electrode. The main body of the
box, where the gel is placed, is filled with a salt-containing buffer
solution that can conduct current. Although you may not be able to
see in the image above (thanks to my amazing artistic skills), the
buffer fills the gel box to a level where it just barely covers the gel.

The end of the gel with the wells is positioned towards the negative
electrode. The end without wells (towards which the DNA fragments
will migrate) is positioned towards the positive electrode.

How do DNA fragments move through the

gel?
Once the gel is in the box, each of the DNA samples we want to
examine (for instance, each PCR reaction or each restriction-
digested plasmid) is carefully transferred into one of the wells. One
well is reserved for a DNA ladder, a standard reference that
contains DNA fragments of known lengths. Commercial DNA
ladders come in different size ranges, so we would want to pick one
with good "coverage" of the size range of our expected fragments.

Next, the power to the gel box is turned on, and current begins to
flow through the gel. The DNA molecules have a negative charge
because of the phosphate groups in their sugar-phosphate backbone,
so they start moving through the matrix of the gel towards the
positive pole. When the power is turned on and current is passing
through the gel, the gel is said to be running.
DNA samples are loaded into wells at negative electrode end of gel.

Power is turned on and DNA fragments migrate through gel (towards

the positive electrode).

After the gel has run, the fragments are separated by size. The largest
fragments are near the top of the gel (negative electrode, where they
began), and the smallest fragments are near the bottom (positive
electrode).

As the gel runs, shorter pieces of DNA will travel through the pores
of the gel matrix faster than longer ones. After the gel has run for
awhile, the shortest pieces of DNA will be close to the positive end
of the gel, while the longest pieces of DNA will remain near the
wells. Very short pieces of DNA may have run right off the end of
the gel if we left it on for too long (something I've most definitely
been guilty of!).

Visualizing the DNA fragments

Once the fragments have been separated, we can examine the gel
and see what sizes of bands are found on it. When a gel is stained
with a DNA-binding dye and placed under UV light, the DNA
fragments will glow, allowing us to see the DNA present at different
locations along the length of the gel.
The bp next to each number in the ladder indicates how many base pairs long the DNA fragment
is.

A well-defined “line” of DNA on a gel is called a band. Each band

contains a large number of DNA fragments of the same size that
have all traveled as a group to the same position. A single DNA
fragment (or even a small group of DNA fragments) would not be
visible by itself on a gel.

By comparing the bands in a sample to the DNA ladder, we can

determine their approximate sizes. For instance, the bright band on
the gel above is roughly 700700700 base pairs (bp) in size.
 Leftmost lane: ladder with 3000 bp, 1500 bp, and 500 bp bands
marked on it.

Lane 1: 5000 bp band.

Lane 2: 100 bp band.

Lane 3: 1500 bp and 2000 bp bands.

Lane 4: 500 bp band.

Four lanes are numbered on the gel above. (A lane is a corridor

through which DNA passes as it leaves a well.)

Which lane matches each description below?

 Key points:
 DNA sequencing is the process of determining the sequence of
nucleotides (As, Ts, Cs, and Gs) in a piece of DNA.

 In Sanger sequencing, the target DNA is copied many times,

making fragments of different lengths. Fluorescent “chain
terminator” nucleotides mark the ends of the fragments and allow
the sequence to be determined.

 Next-generation sequencing techniques are new, large-scale

approaches that increase the speed and reduce the cost of DNA
sequencing.

What is sequencing?
You may have heard of genomes being sequenced. For instance, the
human genome was completed in 2003, after a many-year,
international effort. But what does it mean to sequence a genome, or
even a small fragment of DNA?

DNA sequencing is the process of determining the sequence of

nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. Today,
with the right equipment and materials, sequencing a short piece of
DNA is relatively straightforward.

Sequencing an entire genome (all of an organism’s DNA) remains a

complex task. It requires breaking the DNA of the genome into
many smaller pieces, sequencing the pieces, and assembling the
sequences into a single long "consensus." However, thanks to new
methods that have been developed over the past two decades,
genome sequencing is now much faster and less expensive than it
was during the Human Genome Project^11start superscript, 1, end
superscript.

In this article, we’ll take a look at methods used for DNA

sequencing. We'll focus on one well-established method, Sanger
sequencing, but we'll also discuss new ("next-generation") methods
that have reduced the cost and accelerated the speed of large-scale
sequencing.

Sanger sequencing: The chain termination

method
Regions of DNA up to about 900900900 base pairs in length are
routinely sequenced using a method called Sanger sequencing or
the chain termination method. Sanger sequencing was developed
by the British biochemist Fred Sanger and his colleagues in 1977.

In the Human Genome Project, Sanger sequencing was used to

determine the sequences of many relatively small fragments of
human DNA. (These fragments weren't necessarily 900900900 bp or
less, but researchers were able to "walk" along each fragment using
multiple rounds of Sanger sequencing.) The fragments were aligned
based on overlapping portions to assemble the sequences of larger
regions of DNA and, eventually, entire chromosomes.

Although genomes are now typically sequenced using other methods

that are faster and less expensive, Sanger sequencing is still in wide
use for the sequencing of individual pieces of DNA, such as
fragments used in DNA cloning or generated through polymerase
chain reaction (PCR).
 Ingredients for Sanger sequencing
Sanger sequencing involves making many copies of a target DNA
region. Its ingredients are similar to those needed for DNA
replication in an organism, or for polymerase chain reaction (PCR),
which copies DNA in vitro. They include:

 A DNA polymerase enzyme

 A primer, which is a short piece of single-stranded DNA that binds

to the template DNA and acts as a "starter" for the polymerase

 The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)

 The template DNA to be sequenced

However, a Sanger sequencing reaction also contains a unique

ingredient:

 Dideoxy, or chain-terminating, versions of all four nucleotides

(ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different
color of dye

Dideoxy nucleotides are similar to regular, or deoxy, nucleotides,

but with one key difference: they lack a hydroxyl group on the 3’
carbon of the sugar ring. In a regular nucleotide, the 3’ hydroxyl
group acts as a “hook," allowing a new nucleotide to be added to an
existing chain.

Once a dideoxy nucleotide has been added to the chain, there is no

hydroxyl available and no further nucleotides can be added. The
chain ends with the dideoxy nucleotide, which is marked with a
particular color of dye depending on the base (A, T, C or G) that it
carries.

Method of Sanger sequencing

The DNA sample to be sequenced is combined in a tube with
primer, DNA polymerase, and DNA nucleotides (dATP, dTTP,
dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy
nucleotides are added as well, but in much smaller amounts than the
ordinary nucleotides.

The mixture is first heated to denature the template DNA (separate

the strands), then cooled so that the primer can bind to the single-
stranded template. Once the primer has bound, the temperature is
raised again, allowing DNA polymerase to synthesize new DNA
starting from the primer. DNA polymerase will continue adding
nucleotides to the chain until it happens to add a dideoxy nucleotide
instead of a normal one. At that point, no further nucleotides can be
added, so the strand will end with the dideoxy nucleotide.

This process is repeated in a number of cycles. By the time the

cycling is complete, it’s virtually guaranteed that a dideoxy
nucleotide will have been incorporated at every single position of the
target DNA in at least one reaction. That is, the tube will contain
fragments of different lengths, ending at each of the nucleotide
positions in the original DNA (see figure below). The ends of the
fragments will be labeled with dyes that indicate their final
nucleotide.

Image modified from "Sanger sequencing," by Estevezj (CC BY-SA 3.0). The modified image is
licensed under a (CC BY-SA 3.0) license.

After the reaction is done, the fragments are run through a long, thin
tube containing a gel matrix in a process called capillary gel
electrophoresis. Short fragments move quickly through the pores of
the gel, while long fragments move more slowly. As each fragment
crosses the “finish line” at the end of the tube, it’s illuminated by a
laser, allowing the attached dye to be detected.

The smallest fragment (ending just one nucleotide after the primer)
crosses the finish line first, followed by the next-smallest fragment
(ending two nucleotides after the primer), and so forth. Thus, from
the colors of dyes registered one after another on the detector, the
sequence of the original piece of DNA can be built up one
nucleotide at a time. The data recorded by the detector consist of a
 series of peaks in fluorescence intensity, as shown in
the chromatogram above. The DNA sequence is read from the
peaks in the chromatogram.

Uses and limitations

Sanger sequencing gives high-quality sequence for relatively long
stretches of DNA (up to about 900900900 base pairs). It's typically
used to sequence individual pieces of DNA, such as bacterial
plasmids or DNA copied in PCR.

However, Sanger sequencing is expensive and inefficient for larger-

scale projects, such as the sequencing of an entire genome or
metagenome (the “collective genome” of a microbial community).
For tasks such as these, new, large-scale sequencing techniques are
faster and less expensive.

Next-generation sequencing
The name may sound like Star Trek, but that’s really what it’s
called! The most recent set of DNA sequencing technologies are
collectively referred to as next-generation sequencing.

There are a variety of next-generation sequencing techniques that

use different technologies. However, most share a common set of
features that distinguish them from Sanger sequencing:

 Highly parallel: many sequencing reactions take place at the same

time

 Micro scale: reactions are tiny and many can be done at once on a
chip
 Fast: because reactions are done in parallel, results are ready much
faster

 Low-cost: sequencing a genome is cheaper than with Sanger

sequencing

 Shorter length: reads typically range from 505050 -

700700700 nucleotides in length

Conceptually, next-generation sequencing is kind of like running a

very large number of tiny Sanger sequencing reactions in parallel.
Thanks to this parallelization and small scale, large quantities of
DNA can be sequenced much more quickly and cheaply with next-
generation methods than with Sanger sequencing. For example, in
2001, the cost of sequencing a human genome was
almost \$100$100dollar sign, 100 \text{million}millionstart text, m,
i, l, l, i, o, n, end text. In 2015, it was just \$1245$1245dollar sign,
1245^22squared!

Why does fast and inexpensive sequencing matter? The ability to

routinely sequence genomes opens new possibilities for biology
research and biomedical applications. For example, low-cost
sequencing is a step towards personalized medicine – that is, medical
treatment tailored to an individual's needs, based on the gene
variants in his or her genome.

Questions:

What is the basic purpose of PCR?

How do you specify which DNA to amplify?
Why is Taq polymerase used in PCR?
Why is the DNA heated to94C?
How many cycles of denaturation-hybridization-synthesis are usually conducted for
PCR?
What size fragments can be amplified?
What size primers should you use?
How do you determine the hybridization or annealing temperature for the primers to
attach to the template?
After PCR, what types of procedures are done with the amplified fragments?
What adjustments in the PCR process might you do if you want to clone the PCR
product?
What is the error rate of Taq polymerase?