Chapter 10: BiotechnologyPart 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 COOLHEAT COOL

HEAT COOL

Polymerase Chain Reaction (PCR)

Studying DNA: DNA Fingerprinting

• Like each human has a unique 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 size of the copies of DNA 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 semi-solid 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

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 FingerprintingCriminal Cases Paternity 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.

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• 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, researcher 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• 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.)