Chapter 16 Chapter 16 Recombination DNA and Genetic Recombination DNA and Genetic

EngineeringEngineering

Cloning DNA• 1. Cloning is the production of identical copies• 2. An underground stem sends up new shoots that are

clones• 3. Members of a bacterial colony on a petri dish are

clones because• they all came from division of the same cell.• 4. Human identical twins are clones; the original single

embryo separate to become two individuals.• 5. Gene cloning is production of many identical

copies of the same gene.

Continue…• 6. If the inserted gene is replicated and

expressed, we can recover the cloned gene or protein product.

• 7. Cloned genes have many research purposes: determining the base sequence between normal and

• mutated genes, altering the phenotype, etc.

• 8. Humans can be treated with gene therapy; alteration of other organisms forms transgenic organisms.

Clogged Arteries• Cholesterol does good things for the body,

such as forming membranes and vitamin D, but it can also combine with lipoproteins to form atherosclerotic plaques in the walls of the arteries– Some persons have genes that cause familial

cholesterolemia– Gene therapy promises a way to genetically

alter the cells of the liver to keep the levels of cholesterol in the more normal range

• We can “engineer” genetic changes through recombinant DNA technology.– DNA from different species can be cut,

spliced together, and inserted into bacteria, which then multiply the DNA necessary for protein production

– Genetic engineering has great promise for agriculture, medicine, and industry.

Restriction Enzyme• Bacteria possess restriction enzyme

whose usual function is to cut apart foreign DNA molecules.

• Each enzyme cut only at sites that possess a specific base sequence

• The wide variety of restriction enzymes and their specificity makes it possible to study the genome of a particular species

Modification Enzymes

• Many times the “sticky ends” that result from the cut can be used to pair up with another DNA fragment cut by the same enzyme.

• DNA fragments produced by restriction enzymes are treated with DNA ligase to splice the DNA fragments together to form a recombinant DNA molecule.

Cloning Vectors for Amplifying DNA

• Plasmids are circular DNA molecules in bacteria that carry only a few genes and can replicate independently of the single “main” chromosomes

• When the plasmids is replicated, any foreign DNA that might have become incorporated into it is also replicated producing a DNA clone.

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• Modified plasmids that are capable of accepting, replicating, and delivering DNA to another host cell are called cloning vectors

Reverse Transcriptase to Make cDNA

• Even after a desired gene has been isolated and amplified, it may not be translated into functional protein by the bacteria because introns (noncoding regions) are still present.

• Researchers minimize this problem by using cDNA, which is made from “mature mRNA transcripts.

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– The cDNA is made from mRNA by reverse transcriptase

– cDNA can be inserted into a plasmid for amplification

PCR

• The polymerase chain reaction (PCR) can be used to make millions of copies of cDNA

• 1. PCR can create millions of copies of a single gene or a specific piece of DNA in a test tube.

• 2. PCR is very specific—the targeted DNA sequence can be less than one part in a million of the total DNA sample; therefore a

• single gene can be amplified using PCR.• 3. The polymerase chain reaction (PCR) uses the enzyme

DNA polymerase to carry out multiple replications (a chain reaction) of

• target DNA.• 4. PCR automation is possible because heat-resistant DNA

polymerase from Thermus aquaticus, which grows in hot springs, is an enzyme that withstands the temperature necessary to separate double stranded DNA.

What are Primers?

• Primers are short nucleotide sequences that are made in the laboratory.

• They are recognized by DNA polyermases as the START tags for building complementary sequences of DNA dictated by computer programs stored in the machines.

• DNA FINGERPRINTS ** READ PAGE 257

How is DNA sequenced?• Current laboratories use automated DNA

sequencing to determine the unknown sequence of bases in a DNA sample.

• The automated DNA sequencer separates the sets of fragments by gel electrophoresis.– The “tag” base at the end of each fragment in the

set is identified by laser beam.– The computer program in the machine assembles

the information from all the nucleotides in the sample to reveal the entire DNA sequence

How can you isolate a particular How can you isolate a particular gene for study?gene for study?

• Gene Library: a collection of bacteria that house different cloned DNA fragments– The library of the entire genome or of cDNA is

free of introns

What are Probes?What are Probes?

• DNA probes, short DNA sequences assembled from radioactive nucleotides, can pair with parts of the gene to be studied

• This nucleic acid hybridization technique can be used with other procedures to select cells and their DNA, which may be of interest to the researcher

Analyzing DNA Segments

• 1. Mitochondria DNA sequences in modern living populations can decipher the evolutionary history of human populations.

• 2. DNA fingerprinting is the technique of using DNA fragment lengths, resulting from restriction enzyme cleavage and amplified

• by PCR, to identify particular individuals.• a. DNA is treated with restriction enzymes to cut it into

different sized fragments.• b. During gel electrophoresis, fragments separate

according to length, resulting in a pattern of bands.• .

• c. DNA fingerprinting can identify deceased individuals from skeletal remains, perpetrators of crimes from blood or semen

• samples, and genetic makeup of long-dead individuals or extinct organisms

• 3. PCR amplification and DNA analysis is used to:

• a. detect viral infections, genetic disorders, and

• cancer;

• b. determine the nucleotide sequence of human

• genes: the Human Genome Project; and

• c. associate samples with DNA of parents because it is inherited.

Using the Genetic Strips

• Microorganisms can produce useful substances such as human insulin and blood-clotting factors.

• Genetically engineered bacteria can clean up messes such as oil spills

• Gene may help use devise counterattacks against rapidly mutating pathogens

Transgenic Bacteria• 1. Bacteria are grown in large vats called bioreactors.• a. Foreign genes are inserted and the product is

harvested. (insulin, hepatitis B vaccine, t-PA, and human growth hormone)

• 2. Transgenic bacteria have been produced to protect and improve the health of plants (frost resistance, pesticides)

• 3. Transgenic bacteria can degrade substances (oil-eating bacteria, bio-filters)

• 4. Transgenic bacteria can produce chemical products. (phenylalanine for aspartame sweetener)

Designer Plants (Transgenic Plants)

• Regenerating plants from cultured cells– Botanist are searching the world for seeds from

the wild ancestors of potatoes, corn, etc.• They are worry is that there is too little

diversity in the few strains now used for food crops

– Many plant species can be regenerated from cultured cells.

– Useful mutations, such as resistance to a toxin, can be identified

– Foreign genes now give cotton, corn, and potato strains ability to produce an insect toxin

– Plants are being engineered to produce human proteins including hormones, clotting factors, and antibodies in their seeds; antibodies made by corn deliver radioisotopes to tumor cells and a soybean engineered antibody can treat genital herpes.

How are Genes Transferred Into How are Genes Transferred Into Plants?Plants?

• An early experiment showed that a plasmid from a bacterium that normally causes tumors in plants could be modified by replacing the tumor gene with the desirable genes

Gene Transfer in Animals

• Supermice and Biotech Barnyards– In 1982, the rat gene for somatotropin

production was introduced into mouse eggs; the mice which subsequently expressed the rat gene grew larger than their littermates

– Farm animals may be used to produce TPA for diminishing the severity of heart attacks or CFTR is in the diminishing of cystic fibrous

• 1. Animal use requires methods to insert genes into eggs of animals.

• a. It is possible to micro-inject foreign genes into eggs by hand.

• b. Vortex mixing places eggs in an agitator with DNA and silicon-carbide needles that make tiny holes which the DNA can enter.

• c. Using this technique, many types of animal eggs have been injected with bovine growth hormone (bGH) to produce larger fishes, cows, pigs, rabbits, and sheep.

Gene Pharming Gene pharming is the use of transgenic

farm animals to produce pharmaceuticals; obtainable from the milk of females.

• a. Genes for therapeutic proteins are inserted into animal’s DNA; animal’s milk produces proteins.

• b. Drugs obtained through gene pharming are planned for the treatment of cystic fibrosis, cancer,blood diseases, and other disorders.

Cloning Transgenic Animals• 1. For many years, it was believed that adult

vertebrate animals could not be cloned; the cloning of Dolly in 1997 demonstrated this can be cone.

• 2. Cloning of an adult vertebrate would require that all genes of an adult cell be turned on again.

• 3. Cloning of mammals involves injecting a 2n nucleus adult cell into an enucleated egg.

• 4. The cloned eggs begin development in vitro and are then returned to host mothers until the clones are born.

• 5. Somatic Cell Nuclear Transfer – cloning from adult cells (Dolly)

Animal Organs as Biotechnology Products

• 1. It may be possible to use genetically engineered pigs to serve as a source of organs for human transplant.

• 2. Scientists are learning how to stimulate human cells to construct organs in the laboratory.

Mapping the Human Genome

• Human Genome Initiative is dependent on technology

• The information gained will give insights into genetic disorders and utimately provide gene therapy

• New field of genomics will be concerned with mapping and sequencing the genomes as well understanding the evolutionary relationships

Safety Issues

• Genetically Engineered bacteria have “fail safe” genes included in the DNA which are supposed to be lethal if the bacteria escapes into a non-lab environment

• General public is concerned about organisms being released that are not “natural” and may endanger human lives

Biotechnology in a Brave New World

• Microarray or gene chips can reveal a stunning amount of information about an individual DNA

Gene Therapy

• Has been successful in a trail against SCID-X1

• Eugenic engineering is idea of being able to select desirable humans traits

Send in the Clones

• Xenotransplantation is the transferring of an organ from one species to another

• Pigs can be engineered to lack certain genes that would cause rejection problems when their organs are transplanted to humans

Weighing the Benefits and Risks

• Some say we should never alter the DNA of any organisms; others say we already have

• What would be problems with genetic engineering over the long haul?

• Scenario 1: Melissa is a happy 5 year old who is loved by her family. She becomes ill and is diagnosed with childhood leukemia. A desperate search ensues to find a bone marrow donor whose type matches Melissa. After a year of searching, Melissa’s outlook is grim. Her family decides to clone Melissa so that her clone could be the bone marrow donor. Do you think this is a god idea? Why or why not.

• Scenario 2: A well-loved horse named Barbero breaks his leg in a race. Many people were praying for his well being and thousands of dollars were spent trying to get him to recover. Mail and flowers poured into the animal hospital and stable where Barbero lived. Also, after a year of poor recovery, the decision was made to euthanize Barbero. The owners save sample of his DNA so that Barbero can be cloned. Do you think they should clone him? Why or why not.