Download Chapter 10: Biotechnology

Chapter 10:
Biotechnology
Part 2
DNA Libraries
• The entire set of DNA from an organism-its genome- contains
all of its genes, which generally number in the thousands.
• Scientists must first isolate the gene they are interested in
from the entire genome in order to study and/or manipulate
it.
• To isolate a gene, researchers must first cut the organism’s
genome into pieces using a restriction enzyme.
• Then they clone (copy) all of the pieces by inserting them into
plasmids, which are then taken up by bacteria.
• The bacteria then copy themselves over and over.
• What results from this process is a genomic library, a set of
bacterial clones that collectively contain all of the DNA/genes
in an organism’s genome.
DNA Libraries
DNA Libraries
• So, how do scientists isolate a single gene from an organism’s DNA
library?
• Scientists use a radioactive-labelled probe to isolate their gene of
interest.
• A probe is a fragment of DNA labeled with a tracer (a radioactive
marker such as a radioactive phosphate group).
• The probe is designed to have a nucleotide sequence that is
complementary to the gene that scientists want to isolate.
• This allows the probe to base-pair (or hybridize) with the gene of
interest.
• This is called nucleic acid hybridization and just refers to the fact
that DNA from more than one source has base-paired with each
other.
• This process allows scientists to pinpoint a specific gene of interest
by detecting the radioactive tracer on the probe.
Nucleic Acid Hybridization
Polymerase Chain Reaction (PCR)
• PCR (Polymerase Chain Reaction) is a technique used to produce
thousands or more copies of DNA from a very small sample
without having to clone it using vectors such as plasmids.
• The starting material for PCR is a small sample of DNA with the
desired sequence or gene in it.
• The DNA used to perform PCR may come from cloned bacteria, a
sperm, a mummy, or a single hair root from a crime scene. In fact,
any sample of DNA can be used to do PCR.
Polymerase Chain Reaction (PCR)
• First, the DNA sample is mixed with DNA polymerase, free DNA
nucleotides, and primers.
• Primers are short, single-stranded pieces of DNA that can base-pair
with the desired DNA sequence.
• Next, this mixture is taken through repeated cycles of high and low
temperatures.
• The high temperatures denature the DNA, disrupting the hydrogen
bonds between nucleotides, which is what holds the two strands of
DNA.
• Therefore, the high temperatures unwind the DNA and separate the
two strands.
• Then , during the low temperature phase, the single-stranded DNA
hybridizes with complementary partner strands that have been
formed due to the action of a specific DNA polymerase called Taq
polymerase.
Polymerase Chain Reaction (PCR)
• Most DNA polymerases will not function at the very high
temperatures that denature the DNA. (The very high
temperatures would denature the DNA polymerase as well.)
• However, Taq polymerase is a special DNA polymerase that is
taken from a bacterium that lives in hot springs and
hydrothermal vents, called Thermus aquaticus.
• Because Thermus aquaticus is found in very hot environments,
enzymes (such as DNA polymerase) that are taken from this
bacterium are heat-tolerant.
• Taq polymerase builds complementary strands to hybridize
with the single-stranded DNA when it recognizes primers that
have bound to the single-stranded DNA and begins to build
complementary strands at these primers.
Polymerase Chain Reaction (PCR)
• With every cycle of heating and cooling, this same process
happens, so that the amount of DNA is doubled with every
cycle.
• Thirty PCR cycles may amplify the number of template DNA
molecules a billionfold!!!!!
Polymerase Chain Reaction (PCR)
HEAT
HEAT
COOL
COOL
HEAT
COOL
Polymerase Chain Reaction (PCR)
Homework 2: Vocabulary
1. Define:
•
•
•
•
•
•
•
•
•
Primer
Genome
Polymerase chain reaction
Genomic library
Taq polymerase
Probe
Tracer
Nucleic acid hybridization
Thermus aquaticus
1. For each term above, tell whether it is related to creating
DNA libraries or to performing PCR.
Studying DNA: DNA Fingerprinting
• Like each human has a unique fingerprint that can be used to
identify him or her, so do we all have a unique DNA fingerprint
that can be used to identify us.
• Why do we all have a unique DNA fingerprint and how can it
be generated and used to identify an individual?
Why do we all have a unique DNA fingerprint?
• Even though the DNA in every human is 99% the same, there
are differences in that other 1%.
• It is the difference in this 1% that can be used to generate a
DNA fingerprint that can be used to identify an individual.
• We know that every human has a unique DNA sequence, but
what is it that makes it unique when all of our bodies need the
same genes to produce the same proteins to cause our bodies
to function normally?
• The 1% of our DNA that makes it unique from every other
person’s DNA are unique sequences sprinkled throughout our
genome known as short tandem repeats.
• Short tandem repeats are many copies of the same short DNA
sequence (2 to 10 base pairs long), positioned one right after
the other along the length of a chromosome.
Why do we all have a unique DNA fingerprint?
• These short repetitive sequences slip spontaneously into DNA
during DNA replication, the amount of each can grow or shrink
over successive generations.
• For example, one short tandem repeat may be TTTTC.
• One person may have this sequence repeated 15 times in a
certain strand of their DNA, while another person may have
this same sequence, but only repeated twice in this same
strand of DNA.
• One person could have 10 repeats of of CGG, while another
person may have this same sequence repeated 50 times in the
same place in their DNA.
How can a DNA fingerprint be generated and
how can it be used to identify an individual?
• DNA fingerprinting reveals differences in the number of
tandem repeats among different individuals’ DNA.
• First, PCR is used to make many copies of a region of a
chromosome known to have tandem repeats.
• The a restriction enzyme is used to cut the DNA copies into
fragments.
• The size of the fragments of DNA produced by the restriction
enzyme differs among different individuals because the
number of tandem repeats each person has also differs.
• These differences in the number of tandem repeats can be
detected by gel electrophoresis as differences in the sizes of
fragments generated from restriction enzyme cleavage of a
sample of DNA.
How can a DNA fingerprint be generated and
how can it be used to identify an individual?
• Specifically, gel electrophoresis is a process in which an electric field pulls
different sized DNA fragments (generated by cleavage of the DNA with a
restriction enzyme) through a semi-solid gel.
• DNA fragments of different sizes move through the gel at different rates.
• Smaller fragments will move through a gel much further in a given period of
time than will larger fragments.
• In other words, the smaller the fragment, the faster it moves because shorter
fragments have an easier time slipping between the molecules of the semisolid gel.
• This difference in the speed at which different sized fragments move through
the gel results in a banding pattern of the DNA within the gel that is unique to
every individual- their DNA fingerprint.
• And since we all have different numbers of tandem repeats at specific locations
in our DNA, everyone’s DNA will produce different sized fragments when cut
with a restriction enzyme and will therefore produce a unique banding pattern
when analyzed by gel electrophoresis.
Gel Electrophoresis
A DNA Fingerprint: Homework 3
Explain what caused the banding pattern in each lane to be different from the others.
DNA Fingerprinting
• For all practical purposes, each individual’s DNA fingerprint is unique
to that individual.
• In fact, except for identical twins, the chances that any two people
would have identical tandem repeats in even three regions of DNA is 1
in 1,000,000,000,000,000,000. This is one in a quintillion, which is
much more than the number of people that are even alive on Earth!!!
• Usually, a standard set of thirteen short tandem repeat regions is used
to make a DNA fingerprint of an individual if it is to be used in any
court in the U.S.
• There are many applications for this technology including paternity
disputes, criminal cases, and identifying the remains of individuals
(This technique was used to identify remains from the World Trade
Center tragedy that occurred on September 11th, 2001.).
• It can also be used to determine genetic relationships among
individuals and to trace an individual’s ethnic heritage.
Applications of DNA Fingerprinting
Homework 4
Criminal Cases
Identify the guilty suspect from the first picture. Identify
the father of the child from the second picture. How were
you able to determine this information? Explain.
Paternity Cases
Studying DNA:
The Human Genome Project
• The Human Genome Project involved sequencing the entire
human genome.
• Sequencing refers to a method of determining the order (or
sequence) of nitrogen bases in a fragment of DNA. However,
in this case, we were wanting to know the sequence of every
nitrogen base in human DNA.
• Researchers realized that this project could have enormous
payoffs in medicine and research.
• There was somewhat of a race between private and public
sectors to finish the sequencing of the entire human genome
first.
Studying DNA:
The Human Genome Project
• Private sector companies planned to patent the human genome
and commercialize human genetic information in order to profit
from the information.
• Private companies began trying to sequence the human genome in
1987.
• Walter Gilbert, one of the early inventors of DNA sequencing,
began his own private company and intended to sequence and
patent the human genome.
• This incited the public sector to get involved in sequencing the
human genome.
• In 1988, the National institutes of Health (NIH) hired James
Watson (remember him?) to head the official Human Genome
Project.
• Watson set aside 3% of his $200 million a year budget for the
study of ethical and social issues that would arise from this
research.
• Later, geneticist Francis Collins took Watson’s place.
Studying DNA:
The Human Genome Project
• In 1998, Celera Genomics and its head, Craig Venter, intended to
sequence the human genome, patent its genetic information, and
commercialize the information.
• This prompted the public sector to work even harder and faster to
complete the Human Genome Project.
• In 2000, U.S. President Bill Clinton and British Prime Minister Tony
Blair declared that the sequence of the human genome could not be
patented.
• By 2001, 90% of the sequence of the human genome had been
published.
• In 2003, the Human Genome Project was officially completed.
• In 2010, about 99% of the coding regions of human DNA had been
identified, yet it was not known exactly what every coding region
coded for.
• The next step is to find out what every coding sequence means.
The Human Genome Project
The Human Genome:
Homework 5
• Do you agree with President Clinton’s and British Prime
Minister Blair’s declaration that the human genome could not
be patented? Why or why not?
• How might you have personally been affected if the
information from the Human Genome Project had been
patented and commercialized?
Studying DNA: Genomics
• Genomics is the convergence of investigations into the genomes of
humans and other species.
• Structural genomics focuses on determining the 3-D structure of
the proteins encoded by a genome.
• Comparative genomics compares the genomes of different species
because similarities and difference reflect evolutionary
relationships.
• Also, we are able to decipher the human genome sequence by
comparing it to the genomes of other organisms.
• The premise behind this is that all organisms are descended from
shared ancestors so that all genomes are related to some extent.
• This is evident when comparing sequence data.
• For example, human and mouse DNA sequences are about 78%
identical.
• Human and banana DNA sequences are about 50% identical.
• This tells us we are more closely related to a mouse than to a
banana.
Studying DNA: Genomics
• However, gene-by-gene analyses have many more practical benefits.
• In fact, we have learned the function of many human genes by studying
their counterparts in other species.
• For example, researchers might learn the function of a certain gene in
humans by disabling the expression of that same gene in a mouse and
then observing the consequences.
• This disabling of the expression of a gene in an organism to identify the
function of that gene is called gene knockout.
• For example, researchers discovered the function of the human version of
the mouse APOA5 gene by knocking out this gene in mice.
• This gene encodes a protein that helps to transport dietary fats in the
blood.
• Mice with an APOA5 knockout have four times the normal level of
triglycerides in their blood.
• Researchers then looked for and found a correlation between human
APOA5 mutations and high triglycerides.
• This discovery has medical implications: Individuals with these mutations
have a higher risk for coronary artery disease.
Studying DNA: Genomics
Genomics: Homework 6
• Explain how scientists used the mouse neuroligin 3 gene to
study this gene’s function in children with autism. (See p. 193
in your text.)