Campbell Essential Biology, Seventh Edition,
Global Edition and Campbell Essential Biology
with Physiology, Sixth Edition, Global Edition
Chapter 12
DNA Technology
PowerPoint® Lectures created by Edward J. Zalisko, Eric J. Simon, Jean L. Dickey, and
Jane B. Reece
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DNA Profiling and Forensic Science
• DNA profiling
– is the analysis of DNA samples to determine whether they come from the same
individual and
– has rapidly transformed the field of forensics—the scientific analysis of evidence
for crime scene investigations and other legal proceedings.
• To produce a DNA profile, scientists compare sequences in the genome that vary from
person to person. Figure 12.12 presents an overview of a typical investigation using
DNA profiling.
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Overview of DNA Profiling
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DNA Profiling Techniques
The Polymerase Chain Reaction
• The polymerase chain reaction (PCR)
– is a technique by which a specific segment of DNA can be amplified (by targeting
and copying it quickly and precisely) and
– permits a scientist to obtain enough DNA from even minute amounts of blood or
other tissue to allow a DNA profile to be constructed.
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DNA Amplification by PCR
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The Polymerase Chain Reaction (1 of 2)
• A DNA molecule within a starting sample is likely to be very long. But, most often, only a
very small target region of that large DNA molecule needs to be amplified.
– The key to amplifying one particular segment of DNA and no others is the use of
primers, short (usually 15–20 nucleotides long), chemically synthesized single-
stranded DNA molecules.
– The primers bind to sequences that flank the target sequence, marking the start
and end points for the segment of DNA to be amplified.
• In addition to forensic applications, PCR can be used in the treatment and diagnosis of
disease. PCR can be used to
– amplify, and thus detect, HIV in blood or tissue samples and
– diagnose hundreds of human genetic disorders by being used with primers that
target the genes associated with these disorders.
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Short Tandem Repeat Analysis (1 of 2)
• How do you prove that two samples of DNA come from the same person?
– Repetitive DNA makes up much of the DNA that lies between genes in humans
and consists of nucleotide sequences that are present in multiple copies in the
genome.
– Short tandem repeats (STRs) are short sequences of DNA repeated many
times, tandemly (one after another), in the genome.
– STR analysis is a method of DNA profiling that compares the lengths of STR
sequences at specific sites in the genome.
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Short Tandem Repeat Analysis (2 of 2)
• Consider the two samples of DNA in Figure 12.14.
– Imagine that the top DNA segment was from a crime scene and the bottom from a
suspect’s blood.
– The two segments have the same number of repeats at the first site: 7 repeats of
the four-nucleotide DNA sequence AGAT (in orange).
– However, there isn’t a match at the second site.
– To create a DNA profile, a scientist uses PCR to specifically amplify the regions of
DNA that include these STR sites. The resulting fragments are then compared. In
this case, the comparison reveals that the two samples were not from the same
person.
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Short Tandem Repeat Analysis
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Gel Electrophoresis (1 of 3)
• DNA profiling by STR analysis depends upon comparing lengths of DNA fragments.
This can be done by using gel electrophoresis, a method for sorting
macromolecules—usually proteins or nucleic acids.
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Gel Electrophoresis of DNA Molecules
Figure 12.15 shows how gel electrophoresis
separates DNA fragments from different
sources.
Figure 12.16 shows the gel that would result
from using gel electrophoresis to separate the
DNA fragments from the example in Figure
12.14. This figure simplifies the process. An
actual STR analysis uses more than two sites.
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Visualizing STR Fragment Patterns
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Gel Electrophoresis (2 of 3)
• DNA profiling can provide evidence of guilt or innocence. As of 2017, lawyers at the
Innocence Project have helped to exonerate more than 350 convicted criminals,
including 20 on death row.
– The average sentence served by those who were exonerated was 14 years.
– In nearly half of these cases, DNA profiling has also identified the true
perpetrators.
– Figure 12.17 presents some data from a real case in which STR analysis proved
a convicted man innocent and helped identify the true perpetrator.
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Gel Electrophoresis (3 of 3)
• In forensic cases using STR analysis with the 13 standard markers, the probability of
two people having identical DNA profiles is somewhere between one chance in 10
billion and one in several trillion.
• Thus, despite problems that can still arise from insufficient data, human error, or flawed
evidence, genetic profiles are now accepted as compelling evidence by legal experts
and scientists alike.
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Investigating Murder, Paternity, an Ancient
DNA
• DNA profiling can be used to
– identify murder victims (including more than
half of the victims of the 2001 Word Trade
Center attack);
– settle a question of paternity
§ proving that Thomas Jefferson or a close
male relative fathered a child with an
enslaved woman, Sally Hemings, and
§ that no heirs of Marie Antoinette survived
the French Revolution (Figure 12.18); and
– help protect endangered species by proving the
origin of contraband animal products.
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Bioinformatics: DNA Sequencing
• In the past decade, new experimental techniques have generated enormous volumes of
data related to DNA sequences.
– The need to make sense of an ever-increasing flood of information has produced
bioinformatics, the application of computational methods to the storage and
analysis of biological data.
– Researchers can exploit the principle of complementary base pairing to determine
the complete nucleotide sequence of a DNA molecule, known as DNA
sequencing.
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DNA Sequencing_Sanger sequencing
- Regions of DNA up to about 900 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. 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 PCR.
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DNA Sequencing_Sanger sequencing
Sanger sequencing involves making many copies of a target DNA region.
•A primer
•The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
•The template DNA to be sequenced
•Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP,
ddGTP), each labeled with a different color of dye
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DNA Sequencing
• In one standard procedure, called “next-generation sequencing (NGS),” DNA is cut
into fragments of around 300 nucleotides, and then thousands or hundreds of
thousands of these fragments are sequenced simultaneously. This technology is rapid
and inexpensive, making it possible to sequence more than 2 billion nucleotides in one
day!
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• More recently, scientists have improved or replaced next-generation sequencing.
Several groups have been working on “third-generation sequencing,” wherein a
single, very long DNA molecule is sequenced on its own.
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Genomics
• Genomics is the study of complete sets
of genes (genomes).
– The first targets of genomics
research were bacteria, with
relatively little DNA.
– The genomes of thousands of
species have been published and
tens of thousands are in progress.
– Genome sequences have been
determined for cells from several
cancers, for ancient humans, and
for the many bacteria that live in
the human intestine.
– As the repository of the genetic
information from which all of life’s
inherited characteristics develop,
genomes hold the key to our
genetic identity.
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Comparing Whole Genomes
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Identifying Major Themes (3 of 3)
• Studying whole genomes reveals how genes from one generation can affect the
appearance of the next.
• Which major theme is illustrated by this action?
1. The relationship of structure to function
2. Information flow
3. Pathways that transform energy and matter
4. Interactions within biological systems
5. Evolution
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Genome-Mapping Techniques (1 of 2)
• Genomes are most often sequenced using a
technique called the whole-genome shotgun
method.
– The first step in this method is to chop
the entire genome into fragments using
restriction enzymes.
– Next, all the fragments are cloned and
sequenced.
– Finally, computers running specialized
mapping software reassemble the
millions of overlapping short sequences
into a single continuous sequence for
every chromosome—an entire genome.
– Figure 12.21 summarizes this process.
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Genome-Mapping Techniques (2 of 2)
• The DNA sequences determined by many research groups in the United States are
deposited in GenBank, a database that is available to anyone through the Internet.
– You can browse it yourself at the website for the National Center for Biotechnology
Information: [Link].
– The database is constantly updated, and the amount of data it contains doubles
every 18 months.
– Bioinformatics has revolutionized evolutionary biology by opening a vast new
reservoir of data that can test evolutionary hypotheses
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The Human Genome
• The Human Genome Project was a massive scientific
endeavor to determine the nucleotide sequence of all the
DNA in the human genome and identify the location and
sequence of every gene.
– It started in 1990 and was completed in 2003.
– It remains the world's largest collaborative biological
project
– At the end of the project, more than 99% of the
genome had been determined to 99.999% accuracy.
– This ambitious project has provided a wealth of data
that may illuminate the genetic basis of what it means
to be human. Logo of the Human Genome Project
– The biggest surprise from the Human Genome
Project is the relatively small number of human
genes—currently estimated to be about 21,000.
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The Human Genome
• Like the genomes of most complex eukaryotes, only a small amount of total human
DNA consists of genes that code for proteins, tRNAs, or rRNAs.
– Most complex eukaryotes have a huge amount of noncoding DNA—about 98.5%
of DNA in humans.
– Some of this noncoding DNA is made up of gene control sequences such as
promoters, enhancers, and microRNAs or introns and repetitive DNA.
– Some noncoding DNA is important to our health, with certain regions known to
carry disease-causing mutations. But the function of most noncoding DNA remains
unknown.
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The Human Genome
• The human genome sequenced by government-funded scientists was actually a
reference genome compiled from a group of individuals.
– As of today, the complete genomes of many individuals have been completed.
– We are rapidly approaching the day when an individual’s genome can be
sequenced in a matter of hours for less than $1,000.
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The Human Genome
• Bioinformatics can also provide insights into our evolutionary relationships with
nonhuman animals.
– In 2005, researchers completed the genome sequence for our closest living
relative, the chimpanzee (Pan troglodytes). Comparisons with humans showed
that we share 96% of our genome.
– By comparing humans with related species both living (chimpanzees) and extinct
(Neanderthals), researchers are shedding light on the recent evolutionary history
of our own species and the age-old question of what makes us human.
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The Human Genome
• The potential benefits of knowing many human genomes are enormous. Thus far, more
than 2,000 disease-associated genes have been identified.
– Researchers have long known that this disease is found most commonly among
people living along the ancient trade route in Asia called the Silk Road.
– Researchers discovered four regions of the genome that are associated with the
disease.
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The Process of Science: Did Nic Have a
Deadly Gene (1 of 3)
• Background: When infant Nic Volker stopped breast feeding, he became a medical
mystery.
– Although previously healthy, he now cried in agony after meals and began to waste
away.
– He developed ulcers between his intestines and skin and had to be fed through a
nasal tube.
– After two years of mistaken diagnoses and 100 surgeries, everyone was
desperate.
– Doctors hypothesized that Nic had a rare mutation that made his immune system
attack his digestive system. But how could they prove that idea?
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The Process of Science: Did Nic Have a
Deadly Gene (2 of 3)
• Method: A team of doctors sequenced Nic’s entire protein-coding genome.
– When doctors compared Nic’s genome with other sequenced human genomes,
they found over 16,000 mutations, the vast majority of which were not medically
relevant.
– They developed software to filter the mutations, eliminating ones that did not lead
to malfunctioning proteins or did not involve the digestion or immune systems, and
reduced the list to rare mutations.
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The Process of Science: Did Nic Have a
Deadly Gene (3 of 3)
• Results: After eliminating thousands of mutations, the XIAP gene remained. Nic had a
single base substitution that produced a tyrosine amino acid where cysteine should be in
the protein.
– Data from several species confirmed that cysteine is always at that key location on
the XIAP protein.
– The diagnosis allowed the team to save Nic’s life by replacing his bone marrow
with donor cells that contained functional XIAP genes.
– Today, similar procedures are using personal genome sequencing to save the lives
of many children with rare mutations.
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Applied Genomics
• There are many examples of applied genomics.
– Sequence data provided strong evidence that a Florida dentist transmitted HIV to
several patients,
– that a single natural strain of West Nile virus can infect birds and people, and
– that all of the anthrax spores mailed in a 2001 terrorist attack came from a single
flask at the U.S. Army Medical Research Institute of Infectious Diseases in Fort
Detrick, Maryland.
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Copyright
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