Frontiers of Biotechnology

Manipulating DNA Biotechnology is used to identify people,

produce transgenic organisms and clones, study diseases and evolution, and create medical treatments for people with life threatening diseases.

Techniques to Manipulate DNA Scientists must be

able to work with DNA without being able to see or handle it directly. Scientists use artificial

nucleotides, artificial genes, chemical mutagens, computers, enzymes, bacteria, and many other techniques to manipulate DNA.

Spooled DNA

Restriction Enzymes Restriction enzymes are enzymes that cut DNA molecules at

specific nucleotide sequences. Any time the restriction enzyme comes across the specific

nucleotide sequence, it cuts the DNA. This allows scientists to work with small pieces of DNA at a time.

Restriction enzymes are produced naturally by bacteria to cut the DNA of invading viruses.

Restriction enzymes can either make clean cuts (blunt ends) of the DNA or leave “sticky ends” The “sticky ends” are staggered cuts in the DNA, that allow the

DNA to reform easily.

Restriction Maps Gel Electrophoresis

Once DNA has been cut by restriction enzymes, it is placed into a gel electrophoresis plate. Gel electrophoresis uses an

electrical current to separate a mixture of DNA fragments from each other.

A positive electrode is set at one end, and a negative electrode is set at the opposite end. Because DNA has a negative

charge, the fragments move toward the positive end.

The larger fragments move slower than the smaller fragments, therefore the length of the DNA fragment can be estimated by the distance it travels through the gel in a certain period of time.

Restriction Maps Restriction maps use

gel electrophoresis to show the lengths of DNA fragments between restriction sites in a strand of DNA. Restriction maps are

used to study mutations. They will show if

nucleotides have been added or deleted from a particular strand of DNA.

Or, a mutation may lead to a restriction site, and the DNA would not be cut in the same place.

Copying DNA Forensic scientists use DNA from

cells in a single hair at a crime scene to identify a criminal.

Doctor’s test a patient’s blood to quickly detect the presence of bacteria that causes Lyme disease.

Scientists compare DNA from different species to determine how closely the species are related. If the original DNA from any of

these sources is too small to accurately study, the samples of DNA must be increased, or amplified, so that they can be analyzed.

Polymerase Chain Reaction PCR is a technique that produces millions—or even billions

—of copies of a specific DNA sequence in just a few hours. It is a very simple process. There are four materials involved: the DNA to be copied, DNA

polymerases, large amounts of each of the four nucleotides (A, T, C, G), and two primers. A primer is a short segment of DNA that acts as the starting point for

a new strand. Each PCR cycle doubles the number of DNA copies. The original

piece of DNA becomes two copies. Those two copies become four…ect.

PCR Process1. Separating.

The container with all the reactants is heated to separate the double-stranded DNA into single strands.

2. Binding. The container is cooled and the

primers bind to their complimentary DNA sequences. One primer binds to each DNA strand. The primers bind on the opposite ends of the DNA segment being copied.

3. Copying. The container is heated again and

the polymerases begin to build new strands of DNA. Added nucleotides bind to the original DNA strands by complimentary base pairing. The polymerases continue attaching nucleotides until the entire DNA segment has been copied.

DNA Fingerprinting DNA evidence is used to convict a criminal, release

an innocent person from prison, or solve a mystery. A couple of decades ago, the lines and swirls of

someone’s fingertip were a detective’s best hope for identifying someone. Now, investigators gather biological samples and analyze DNA for another kind of evidence: a DNA fingerprint.

DNA Fingerprinting A DNA fingerprint is a kind of

restriction map. It is a representation of parts

of an individual’s DNA that can be used to identify a person at a molecular level. People differ greatly in the

number of repeated non-coding sequences of DNA.

DNA fingerprints can also be used to show relationships between family members. The children have similar

DNA fingerprints to each other, but they are not identical. Also, their DNA fingerprints are combinations of the DNA fingerprints of the parents.

DNA Fingerprinting and Identification The reliability of DNA identification

relies on probability. Ex. Suppose that 1 in every 500

people has three copies of the repeat at location A. That means that any person has a 1 in

500 chance of having a matching DNA fingerprint for that region of their chromosome.

Then, suppose that 1 in 90 people has six copies of the repeat sequence at location B, and 1 in 120 people has ten copies of the repeat sequence at location C.

Individual probabilities are multiplied by each other to find total probability. Therefore, when the three separate probabilities are multiplied, suddenly the chance that two people have the same DNA fingerprint is very small. 1/500 x 1/90 x 1/120 = 1/5,400,000 =

1 chance in 5.4 million people.

Uses of DNA Fingerprinting DNA fingerprinting can be

used to convict a criminal or set an innocent person free. The Innocence Project has

successfully released 249 wrongly convicted people from jail using DNA fingerprinting.

DNA fingerprinting can be used to identify familial relationships (paternity).

DNA fingerprinting is also used to study biodiversity in an area, identify genetically engineered crops, and to follow migration patterns of native species.

Larry Fuller spent 18 years in prison, after being wrongfully convicted of aggravated rape in 1981. Fuller was excluded as the rapist after advanced DNA testing. He was released in January 2007.

Genetic Engineering Glowing mice are used in

cancer research, glowing plants are used to track genetically modified crops, and in 1999, British researchers introduced glowing yeast cells. The glowing yeast cells can

detect pollution in an environment. Under normal conditions, the cells

do not glow, but when they come into contact with certain chemicals, they glow. This indicated areas that need to be

cleaned up.

Cloning A clone is a genetically identical

copy of a gene or of an organism. Cloning can be a natural process.

Plants can clone themselves, bacteria produce genetically identical copies of themselves, and identical twins are clones of each other.

To clone a mammal, scientists swap DNA between cells with a technique called nuclear transfer. 1. An unfertilized egg is taken from an

animal, and the egg’s nucleus is removed.

2. The nucleus of a cell from the animal to be cloned is implanted into the egg.

3. The egg is stimulated, and if the transfer is successful, the egg will begin dividing.

Dolly: The First Clone Born July 5th, 1996 in

Endinburgh, Scotland—was the fist cloned mammal (cloned from a mammary gland, and named after Dolly Parton) She was the only lamb who

survived to adulthood, out of 227 attempts.

She gave birth to 6 lambs, and died at the age of 6 due to lung disease.

The Future of Cloning In January 2009, scientists in

Spain successfully cloned a Pyrenean Ibex—a species declared extinct in 2000. They used skin cells preserved in

liquid nitrogen. The Ibex died shortly after birth

due to physical defects in its lungs. The possibility of cloning other

endangered or extinct species (like the wooly mammoth or dinosaurs) is closer. This still does not increase genetic

diversity in breeding pools however, and does not help loss of habitat.

Genetic Engineering Genetic research relies on

cloning, but not of entire organisms. Instead, the cloning of

individual genes is used to make a copy of one segment of DNA.

In some cases, scientists insert cloned genes from one organism into an entirely different organism.

Genetic engineering is when you change an organism’s DNA to give an organism new traits. Genetic engineering is only

possible because the genetic code is shared by all organisms.

Recombinant DNA Recombinant DNA is DNA that

contains genes from more than one organism. To create recombinant DNA, scientists

often used plasmids located in bacteria. Plasmids are closed loops of DNA

within the bacteria that are easily manipulated.

Scientists are using recombinant DNA to: Try to create crop plants that produce

medicines and vitamins. Try to create vaccines against HIV. Make hormones like HGH, insulin, and

oxytocin. Use in gene therapy.

ETC!

Transgenic Organisms A transgenic organism has one or more genes

from another organism inserted into the genome. Ex. The gene for human insulin can be put into

plasmids. The plasmids are inserted into bacteria. The transgenic bacteria make human insulin which can be collected and used to treat people with diabetes.

Transgenic Plants Scientists create transgenic

plants by inserting a gene into a bacteria’s plasmid and having the bacteria infect a plant. The infected plant will then incorporate the new gene into its DNA. This technique has allowed

scientists to give plants new traits, such as resistance to frost, diseases, and insects. This increases crop yields,

more food is produced more quickly and cheaply.

Genetically modified (GM) foods, are now common in the United States.

Transgenic Animals Transgenic animals are difficult to make. It take many trials (hundreds)

before a transgenic animal will form correctly to adulthood. The advantage with transgenic animals is that the transgenic gene will be present

in ALL of their DNA, including in their reproductive cell. So, transgenic traits will be passed on to the next generation.

Transgenic mice are often used as models to study human development and disease. They are used to study cancer (oncomice), diabetes, brain function and

development, sex determination, etc. Other mice are called “gene knockout” mice.

These mice are used to student gene functions and point mutations.

Concerns About Genetic Engineering There are concerns regarding human health and

the environment. People routinely eat GM foods without knowing it.

Scientists have not been able to discover any adverse health effects so far. Critics claim that not enough research has been done on

possible allergic reactions or other unknown side effects. Another concern is that GM plants may cause bees and

butterflies to go extinct (by transgenically producing pesticides). Transgenic plants may also cross-pollinate with wild natural

plants. Finally, transgenic plants may decrease genetic diversity in

crops and leave them more vulnerable to new diseases or pests.

Another concern is about the ethics of genetic engineering in the first place.

Genomics Genomics is the study of

genomes, which can include the sequencing of all of an organism’s DNA.

This is how we know that humans and chimpanzees share 99-98% of their DNA.

Comparing DNA from many people at one time helps researchers find genes that cause disease, and it helps them understand how medication works.

Biologists study DNA of different species to learn how closely related they are to each other and to extrapolate how far back in the evolutionary time line they diverged.

DNA Sequencing All genomic studies begin with DNA sequencing.

This is determining the order of DNA nucleotides in genes or in genomes. PCR is one method.

Humans do not have the largest genome. Vanilla plants, crested newts, and lungfish are among the many organisms who have a larger genome than us!

The Human Genome Project There are 30,000-40,000 genes in the

human genome. Each gene represents about 100,000

DNA bases. The Human Genome Project has two

goals:1. To map and sequence all of the DNA

base pairs of the human chromsomes.

2. To identify all of the genes within the sequence. Right now, the Human Genome

Project is working on the HapMap—the study of how DNA sequences vary among people.

This will hopefully identify genetic differences that play a part in human diseases.

Bioinformatics Bioinformatics is the use of computer databases

to organize and analyze biological data. Bioinformatics give scientists a way to store, share,

and find data.

Genetic Screening Genetic screening is the process of testing

DNA to determine a person’s risk of having or passing on a genetic disorder. It often involved both pedigree analysis and DNA

tests. There are tests for about 900 genetic disorders,

including cystic fibrosis, Duchenne’s muscular dystrophy, and breast cancer.

Gene Therapy Gene therapy is the replacement

of a defective or missing gene, or addition of a new gene, into a person’s genome to treat a disease. Scientists will often use de-

natured viruses to introduce the new gene to the body. There have been a few

successful cases of gene therapy wiping out diseases.

There are many experiments going on with gene therapy. Scientists are trying to insert

genes into the immune system that stimulated the immune system to attack cancer cells.

Another method is to insert “suicide” genes into cancer cells.