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CHAPTER 20 – DNA Tools and Biotechnology

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Recombinant DNA molecules are formed when segments of DNA from two different sources—often different species—are combined in vitro.

DNA Technology is the term for analyzing genes and gene expression.

Human lives are greatly affected by biotechnology, the manipulation of organisms or their components to make useful products. - Biotechnology includes such early practices as selective breeding of farm

animals and the use of microorganisms to make wine and cheese. - Today, biotechnology also encompasses genetic engineering, the direct

manipulation of genes for practical purposes.

DNA Toolbox

RECALL: A gene from one organism SHOULD function in any other organism because all organisms have the same GENETIC CODE

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Knowing the entire nucleotide sequence of a gene allows researchers to compare it to genes in other species, whose function may be known.

To study a particular gene, scientists developed methods to isolate the portion of a chromosome that contains the gene of interest.

Techniques for DNA cloning enable scientists to prepare multiple identical copies of well-defined segments of DNA.

In addition, bacteria have plasmids, small circular DNA molecules with a small number of genes that replicate independently from the chromosome.

DNA Cloning

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General CloningOne basic cloning technique begins with the insertion of a “foreign” gene into a bacterial plasmid to produce a recombinant DNA molecule.

The plasmid is returned to a bacterial cell, producing a recombinant bacterium, which reproduces to form a clone of genetically identical cells, (including the genes as well!)

The production of multiple copies of a single gene is called gene cloning.

Gene cloning is useful for two basic purposes: to make many copies of, or amplify, a particular gene and to create a protein product.

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Restriction Enzymes Restriction enzymes cut DNA

at specific recognition points. These splice points are palindromes. They create restriction fragments that can be put into plasmids. They can create two different types of ends:

Blunt Ends – when both ends are paired up

Sticky Ends – when both ends have unpaired nucleotides

DNA Ligase seals the ends.

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Recombinant plasmids are produced when restriction fragments from foreign DNA are spliced into plasmids.

The plasmid is called a cloning vector, a DNA molecule that can carry foreign DNA into a cell and replicate there.

Bacterial plasmids are widely used as cloning vectors because they can be isolated from bacteria, manipulated to form recombinant plasmids by in vitro insertion of foreign DNA, and then introduced into bacterial cells.

Restriction Fragments and Cloning Vectors

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Cloning a Gene using a Plasmid

Procedure:1. Extract plasmid DNA from

bacterial cells and the Gene of interest from eukaryotic DNA

2. Cut plasmid and DNA using the same restriction enzyme

3. Hydrogen bond the plasmid DNA to the DNA fragment you want to insert

4. Use ligase to seal the ends5. Transform the bacteria with the

recombinant plasmidRECALL: Transformation when a cell takes up a plasmid (or a piece of foreign DNA) from the environment and incorporates it into its own genome

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In electrophoresis, the bigger pieces run slower so don’t get as far from the well, the smaller pieces run much faster. The DNA is negatively charged, so it runs to the positive side of the chamber.

Researchers use nucleic acid probes to tag specific segments of DNA (GENE OF INTEREST!) so it is easier to compare the results (otherwise there can be too many bands to tell apart)

After a piece of DNA is digested with restriction enzymes, it can be run on a gel electrophoresis. This technique separates out the fragments of DNA based on their size (length). Different samples can be prepared to look at evidence from a crime scene or paternity tests.

Gel Electrophoresis

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Nucleic Acid ProbesA nucleic acid probe is a single

strand of radioactive DNA or RNA that is the complement of whatever gene they are looking for. The DNA is denatured (the strands are separated) and the probes match up with their complements. The genes of interest can then be identified by the radioactivity.

Nucleic Acid probes are also used in gel electrophoresis. Because gel electrophoresis yields too many bands to distinguish individually, scientists use nucleic acid hybridization with a specific probe to label bands that derive from the gene of interest.

The probe is a radioactive, single-stranded DNA molecule that is complementary to the gene of interest. This allows scientists to identify the fragment they are looking for.

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Southern Blotting Technique

Compares DNA of different individuals.

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PCR

Polymerase Chain Reaction - This is the process of copying pieces of DNA quickly without using live cells.

It can’t replace regular gene cloning for when you need LARGE amounts of the gene.

PCR allows us to make many copies of a targeted segment of DNA.

In PCR, a three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules. It uses special DNA polymerases that can withstand high levels of heat.

PCR has amplified DNA from a variety of sources: fragments of ancient DNA from a 40,000-year-old frozen woolly mammoth; DNA from footprints or tiny amounts of blood or semen found at the scenes of violent crimes; DNA from single embryonic cells for the rapid prenatal diagnosis of genetic disorders; and DNA of viral genes from cells infected with HIV.

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One way around this is to insert an expression vector, a cloning vector containing a highly active bacterial promoter, upstream of the restriction site.

The bacterial host cell recognizes the promoter and proceeds to express the foreign gene that has been linked to it.

Putting EUKARYOTIC genes into PROKARYOTIC

vectors has challengesBacteria CANNOT remove eukaryotic introns so the presence of introns in eukaryotic DNA poses a problem

This problem can be surmounted by using a cDNA (complementary DNA) form of the gene, which includes only the exons.

cDNA is made from the mRNA that has already been processed (and the introns have already been cut out of); the enzyme reverse transcriptase is used to go from mRNA DNA; therefore cDNA does not include any introns.

Molecular biologists can avoid incompatibility problems by using eukaryotic cells (such as yeast - YAC) as hosts for cloning and expressing eukaryotic genes. Yeast are unique because they are eukaryotic, but also have plasmids.

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Electroporation

Electroporation is a process where an electrical stimulus is added to a solution of cells. It creates temporary holes in the cell membrane and allows foreign DNA to enter the cell. Scientists can also use extremely thin needles to introduce the DNA.

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DNA Microarray Assay

This process can detect and measure the expression of thousands of genes at one time. It shows which genes are active and inactive in certain tissues. This is currently being used to determine which genes are expressed in normal cells versus cancer cells. A DNA microarray consists of tiny amounts of a large number of single-

stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array, or grid, also called a DNA chip. Ideally, these fragments represent all the genes of an organism.

The DNA fragments on a microarray are tested for hybridization with cDNA molecules that have been prepared from the mRNAs in particular cells of interest and labeled with fluorescent dyes. In other words, the microarray assay measures what genes are being EXPRESSED (actively transcribed and translated)

DNA microarray assays have a huge impact on genomic studies because they allow the expression of many or even all of the genes in the genome to be compared at once.

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Scientists sometimes introduce mutations to cloned genes to “knock out” the working copy of the gene. By doing this, you can look at the end phenotype and see what difference is caused by the missing protein. This allows researchers to find the function of various genes.

This is not realistic in human genes (ethical issues), so it cannot be used in humans.

Disabling genes to

find function

Researchers carry out large-scale genetic analyses, genome-wide association studies, of large numbers of people with a certain phenotypic condition or disease, such as heart disease or diabetes. They test for genetic markers, DNA sequences that vary in the population.

A single base-pair site where variation is found in at least 1% of the population is called a single nucleotide polymorphism (SNP). A few million SNPs occur in the human genome. SNPs can be detected by very sensitive microarray analysis or by PCR.

FINDING AN SNP THAT INDICATES A SPECIFIC DISORDER Once a region is found that has a SNP shared among affected but not unaffected people, researchers sequence that region. So, in studies that examine a group of humans, they focus on SNP’s where one allele is found more often in people with a specific disorder as compared to people without that disorder. In most cases, the SNP itself does not contribute to the disease. In fact, most SNPs are in noncoding regions.

We have found SNP’s related to diabetes, heart disease, and certain types of cancer so far.

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Cloning

Organismal Cloning Cloning an organism from a single cell; different from gene cloning or cell cloning

Plants have been cloned from the 1950’s because they are totipotent remain flexible in its developmental possibilities; can make the WHOLE plant from one cell

Scientists did a procedure called nuclear transplantation where they removed a nucleus from an UNFERTILIZED frog egg and replaced it with the nucleus from a differentiated cell from a tadpole. If the cell retains its full genetic capacity, it SHOULD be able to direct the development of the egg. What they found is that the nuclear potency (ability to remain flexible in development) decreases as cell differentiation progresses.

Cloning

Dolly (5 July 1996 – 14 February 2003) was a female domestic sheep remarkable in being the first mammal to be cloned from an adult somatic cell, using the process of nuclear transfer. She was cloned at the Roslin Institute near Edinburgh in Scotland. She was born on 5 July 1996 and she lived until the age of six. The cloning of Dolly was considered a major scientific breakthrough because it showed that differentiated adult mammal cells can dedifferentiate and it was also the first successful clone of a mammal.

To produce Dolly, they took the mammary cell of one sheep (ADULT CELL!), and put the cell into G0 (semistarvation). They then took an egg from another sheep and took out the nucleus. They fused the two cells together and cultured it for 6 days. After a period of time, it was implanted into a 3rd sheep (surrogate). Dolly was born in July 1996).

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http://www.nytimes.com/video/us/100000002496111/dolly-the-sheep.html

-Stem cells are unspecialized. They reproduce and under different conditions, differentiate into various cell types. - Most stem cells are found in embryos. Adults have SOME stem cells, mainly located in bone marrow. For example, bone marrow contains several types of stem cells, including one that can generate all the different kinds of blood cells and another that can differentiate into bone, cartilage, fat, muscle, and the linings of blood vessels. -Embryonic stem cells offer more potential than adult stem cells for medical applications, because ES cells are pluripotent, capable of differentiating into different cell types

Stem Cells

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Pluripotent vs. Totipotent

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Gene Therapy

In gene therapy, doctors use bone marrow cells (usually taken from the hip or head of the femur) because they multiply throughout life. They can extract these cells, expose them to a virus carrying a “good” copy of the gene, and then return them to the body.

In gene therapy, doctors can use RETROVIRUSES RNA virus that inserts a DNA copy of their genome into the host cell; in gene therapies we can use the retrovirus to put modified DNA into targeted cells and have that DNA become integrated into the host cells genome

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Pharmaceutical Industry using Biotechnology to create New Drugs

Determining the sequence and structure of proteins crucial for tumor cell survival has led to the identification of small molecules that combat certain cancers by blocking the function of these proteins. Another important pharmaceutical product created by genetic engineering is tissue plasminogen activator (TPA – “clot buster”). If administered shortly after a heart attack, TPA helps dissolve blood clots and reduces the risk of subsequent heart attacks.

DNA cloning and gene expression systems can produce large quantities of proteins that are present naturally in only minute amounts. Among the first pharmaceutical products “manufactured” in this way were human insulin and human growth hormone (HGH).

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Transgenic Organisms

National Taiwan University cloned 3 pigs which are green inside out inclusive their internal organs. Other organisms that have been made that glow are cats, rabbits, and zebrafish.

Transgenic organisms are plants and animals that carry genes from other species. Examples are leaner pigs and more productive cows. They are experimenting with “pharmaceutical factories”… putting a human gene in cows so our protein is in their milk.

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Body fluids or small pieces of tissue may be left at the scene of a violent crime or on the clothes of the victim or assailant.

If enough blood, semen, or tissue is available, forensic laboratories can determine the blood type or tissue type by using antibodies to detect specific cell-surface proteins.

DNA testing, in contrast, can identify the guilty individual with a high degree of certainty because the DNA sequence of every person is unique (except for identical twins).

Genetic markers that vary in the population can be analyzed for a given person to determine that individual’s unique set of genetic markers, or genetic profile.

The FBI started applying DNA technology in forensics in 1988, using RFLP analysis by Southern blotting to detect similarities and differences in DNA samples.

Forensics

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Today, in place of RFLPs, forensic scientists usually use an even more sensitive method, which takes advantage of genetic markers called short tandem repeats (STRs).

STRs are variations in the lengths of certain repeated base sequences in specific regions of the genome. THE NUMBER OF REPEATS PRESENT IN THESE REGIONS IS HIGHLY POLYMORPHIC, VARYING FROM PERSON TO PERSON OR ANIMAL TO ANIMAL.

STR – Short Tandem Repeats

PCR (used to amplify particular STRs) allows this method to be used even when the DNA is in poor condition or available in only minute quantities. In another example of genetic profiling, a comparison of the DNA of a mother, her child, and the purported father can conclusively settle a question of paternity. Analyzing genetic profiles can also identify victims of mass casualties (used in 9/11).

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Microorganisms can be genetically engineered to digest harmful products that have been spilled or leaked into the environment.

Genetically engineered microbes may become important in both mining minerals (especially as ore reserves are depleted) and cleaning up highly toxic mining wastes.

Biotechnologists are also trying to engineer microbes that can be used in wastewater treatment plants to degrade chlorinated hydrocarbon.

Environmental Clean-up

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Plants are easier to genetically engineer than most animals because a single tissue cell grown in culture and genetically manipulated can give rise to an adult plant with new traits.

Genetic engineering is rapidly replacing traditional plant-breeding programs for simple genetic traits such as herbicide or pest resistance.

Genetically engineered crops that can resist destructive insects have reduced the need for chemical insecticides.

Genetically engineered plants include transgenic rice plants that can grow in water of high salinity and also “Golden Rice” which have high levels of Vitamin A.

Agricultural Applications

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A GM organism (genetically modified) is one that has acquired by artificial means one or more genes from another species or even from another variety of the same species.

In the United States, the majority of corn, soybeans, and canola are GM crops, and GM products do not require labeling.

Advocates of a cautious approach toward GM crops fear that transgenic plants might pass their new genes on to close relatives in nearby wild areas.

If crop plants carrying genes for resistance to herbicides, diseases, or insect pests pollinated wild plants, the offspring might become “super weeds” that are very difficult to control.

Organisms used in recombinant DNA experiments are genetically crippled to ensure that they cannot survive outside of the lab.

Ethical Concerns