17: Recombinant DNA and Biotechnology

Introduction

· The use of biotechnology to produce lifesaving drugs, improved crop plants, and even silk-producing goats has come from our knowledge of DNA transcription and translation.

Cleaving and Rejoining DNA

· Recombinant DNA technology is the manipulation and combination of DNA molecules from different sources.

· Recombinant DNA technology uses the techniques of sequencing, rejoining, amplifying, and locating DNA fragments, all of which use complementary base pairing of A with T (or U) and G with C.

A. Restriction endonucleases cleave DNA at specific sequences

· Restriction endonucleases are enzymes that hydrolyze two phosphodiester linkages on opposite strands of double-stranded DNA molecules.

· They recognize and cut specific DNA sequences.

· The sequences recognized by these enzymes are generally short (4 to 6 base pairs) in length.

· Bacteria evolved these enzymes as a defense against viruses. (See Figure 17.1.)

· The site that is cut is called a recognition site or restriction site.

· Bacteria avoid damaging their own DNA by modifying their DNA with methyl groups.

· The enzyme EcoRI cuts double-stranded DNA with the sequence 5¢...GAATTC...3¢.

· Notice that the complementary strand reads the same in the 5¢-to-3¢ direction.

· Pairing up complementary bases with the EcoRI sequence yields CTTAAG, but complementary strands of DNA are antiparallel, so this is reading in the 3¢-to-5¢ direction.

· This type of sequence is called a palindromic sequence.

· The likelihood of randomly matching any given six-base sequence is 1 in 4⁶ (1 in 4,098).

· There are four possibilities for each base, so the total number of possible six-base sequences is 4 ´ 4 ´ 4 ´ 4 ´ 4 ´ 4.

· Using this restriction enzyme on a long stretch of random DNA would create fragments with an average length of 4,098 bases.

· Note that this is the average length for random DNA. Actual lengths will vary widely.

· Using EcoRI to cut up small viral genomes with only a few thousand base pairs may result in only a few fragments.

· For a eukaryotic genome with tens of millions of base pairs, the number of fragments will be very large.

· In nature, DNA is not random. A phage called T7, which has E. coli as a host, has no occurrences of the EcoRI recognition sequence in its 40,000-base-pair genome.

· Hundreds of restriction enzymes have been purified from various organisms, and these enzymes serve as “knives” for genetic surgery.

Gel electrophoresis identifies the sizes of DNA fragments

· The fragments of DNA can be separated using electrophoresis. (See Figure 17.2. The Instructor’s Resource CD-ROM includes a photograph of a gel being examined under ultraviolet light.)

· Because of its phosphate groups, DNA is negatively charged at neutral pH.

· When DNA is placed in a semisolid gel and an electric field (with + and – ends) is applied, the DNA molecules migrate toward the positive pole because opposite charges attract.

· The porous gel acts as a sieve, and smaller molecules can migrate more quickly than larger ones.

· After a fixed time with all fragments on the gel, separated molecules can be examined.

· They may be better visualized by simple staining or by transferring the DNA to a nylon membrane, denaturing the DNA, and using a complementary labeled single-stranded DNA probe. (See Figure 17.3.)

· When double-stranded DNA is heated, it denatures and becomes single-stranded. When cooled, it reanneals, becoming double-stranded. Complementary sequences reanneal.

Recombinant DNA can be made in a test tube

· Some restriction enzymes cut DNA strands bluntly, exactly opposite one another, while others leave staggered ends of single-stranded DNA.

· The enzyme EcoRI leaves staggered ends that are sticky and attract complementaries.

· If two different DNA's are cut so each has EcoRI staggered ends, different fragments with complementary sticky ends can be recombined and sealed with DNA ligase. (See Figure 17.4.)

Cloning Genes

· The goal of recombinant DNA work is to produce many copies (clones) of a particular gene.

· If the DNA is to be used to make protein, it must be introduced, or transfected, into a host cell.

· The host cells are transfected with DNA under special conditions.

· The cells that actually get the DNA must be distinguishable from those that do not.

· This may be done with genetic markers, called reporter genes.

Genes can be inserted into prokaryotic or eukaryotic cells

· Bacteria have been useful as hosts for recombinant DNA.

· Bacteria are easy to manipulate, and they grow and divide quickly (20 to 60 minutes per division).

· They have genetic markers that make it easy to select or screen for insertion.

· They have been intensely studied and much of their molecular biology is known.

· Bacteria have some disadvantages as well.

· Bacteria lack splicing machinery to excise introns.

· Protein modifications, such as glycosylation and phosphorylation, fail to occur as they would in an appropriate eukaryotic cell.

· In some applications, the expression of the new gene in a eukaryote (the creation of a transgenic organism) is the desired outcome.

· Saccharomyces, baker's and brewer's yeast, are commonly used eukaryotic hosts for recombinant DNA studies.

· Yeasts divide quickly for eukaryotic cells (every 2 to 8 hours).

· They are easy to grow.

· Yeasts have relatively small genomes for eukaryotes (about 20 million base pairs).

· Plants are also used as hosts if the goal is to make a transgenic plant.

· It is relatively easy to regenerate an entire plant from differentiated plant cells because of plant cell totipotency.

· The transgenic plant can then reproduce naturally in the field and will carry and express the gene on the recombinant DNA.

Vectors can carry new DNA into host cells

· New DNA can be introduced into the cell's genome by its integration into a chromosome of the host cell.

· New DNA can be incorporated into the host cell by a carrier called a vector.

· A vector has an ability to replicate independently in the host cell.

· A vector must meet the following requirements:

· It must have a recognition sequence for a restriction enzyme, permitting it to form recombinant DNA.

· It must have a genetic marker that will announce its presence in the host cell.

· It should also be small in comparison to host chromosomes.

· Plasmids as vectors:

· Plasmids are ideal vectors for the introduction of recombinant DNA into bacteria.

· A plasmid is a small, circular DNA molecule, separate from the bacterial chromosome. (Chapter 13 of the Instructor’s Resource CD-ROM includes a micrograph of two bacterial plasmids.)

· Plasmids have their own origin of replication and can divide separately from the host bacterium's chromosome.

· They are usually small—2,000 to 6,000 base pairs.

· They often have just one restriction site, if any, for a given restriction enzyme. (See Figure 17.5a.)

· Cutting the plasmid at one site makes it a linear molecule with sticky ends.

· If another DNA is cut with the same enzyme, leaving staggered "sticky" ends, it is possible to insert the DNA into the plasmid. (See Figure 17.4.)

· Plasmids are isolated from bacteria and cut with a restriction enzyme.

· The enzyme is inactivated and the new DNA is added

· DNA ligase is added to seal the plasmids back together with the inserts.

· Plasmids are transferred to a live culture of bacteria that have no plasmids.

· These bacteria are treated so that they will take up the plasmids.

· Plasmids often have selection genes such as antibiotic resistance genes. (See Figure 17.5.)

· Viruses as vectors:

· Only about 5,000 base pairs can be inserted into plasmid DNA.

· For inserting larger DNA sequences, both prokaryotic and eukaryotic viruses are often used as vectors.

· If the genes that cause E. coli host death and lysis (about 200,000 base pairs) are eliminated, the bacteriophage lambda can still infect the host and inject its DNA.

· The deleted 20,000 base pairs can be replaced by DNA from another organism, creating recombinant viral DNA.

· Artificial chromosomes as vectors:

· Bacterial plasmids are not good vectors for yeast (eukaryotic) hosts.

· A yeast artificial chromosome, or YAC, has been made that has a yeast origin of replication, a centromere sequence, and telomeres, making it a true eukaryotic chromosome.

· YACs have been engineered to include specialized single restriction sites and selectable markers. (See Figure 17.5b.)

· YACs are 10,000 base pairs in size, but can accommodate up to 1.5 million base pairs of inserted DNA.

· Human artificial chromosomes (HACs) have been constructed of a human centromere, telomeres, and origins of replication.

· HACs may one day be used as gene therapy vectors.

· Plasmid vectors for plants:

· Plasmid vectors for plants include a plasmid found in the bacteria that causes crown gall, Agrobacterium tumefaciens. (See Figure 17.5c. The Instructor’s Resource CD-ROM includes a photograph of a crown gall and, in Chapter 26, a TEM of A. tumefaciens.)

· Crown gall causes tumors in plants.

· Part of the Ti plasmid of A. tumefaciens is T DNA, a transposon.

· It produces copies of itself in the plant host chromosomes.

· T DNA, tumor-causing sequences, can be replaced by the desired DNA, and the plasmid no longer produces tumors.

· The plant cells that are purposely infected can be used to generate transgenic plants.

There are many ways to insert recombinant DNA into host cells

· DNA usually cannot get across cell membranes.

· High Ca²⁺ causes membrane changes that reduce the barriers to DNA movement.

· Treatment of cells and the DNA with high Ca²⁺ allows DNA uptake by cells.

· Other methods include the following:

· Electroporation—cells are exposed to rapid pulses of high-voltage current to render the plasma membrane permeable.

· Injection—cells are punctured with a tiny pipette, and DNA is inserted through the bore.

· Lipofection—DNA is coated with lipid, which allows it to pass through the plasma membrane.

· Particle bombardments—tiny high-velocity particles of tungsten or gold are coated with DNA and then shot into cells.

Genetic markers identify host cells that contain recombinant DNA

· When a population of host cells is treated to introduce DNA, just a fraction actually incorporate and express it.

· Only a few vectors that move into cells actually contain the new DNA sequence.

· Therefore, a method for selecting for transfected cells and screening for inserts is needed.

· A classic example of how this was originally done is as follows:

· The plasmid pBR322 carries within its sequences an origin of replication and two antibiotic resistance genes: amp^r (ampicillin resistance) and tet^r (tetracycline resistance).

· Within the antibiotic resistance genes are restriction enzyme recognition sites.

· The amp^r gene has a PstI recognition site.

· The tet^r gene has recognition sites for HindIII, BamHI, and SalI.

· In each case these are the only sites for each enzyme in the plasmid.

· If the foreign DNA is cut with BamHI, for example, and the plasmid is too, they can be recombined and sealed together with ligase. (See Figure 17.6.)

· BamHI cuts the plasmid in the gene that codes for resistance to tetracycline, so the resistance to tetracycline is inactivated.

· In practice, when plasmid and foreign DNA are placed together in the test tube, three outcomes are possible:

· Some plasmids just reseal their own ends with no insert being incorporated.

· Some foreign DNA remains free in the solution, without being incorporated into plasmids.

· Some foreign DNA is integrated into plasmids. This is actually the rarest result.

· Bacteria treated with these plasmids might get just the foreign DNA or a plasmid either with or without the insert. (See Figure 17.6.)

· Bacteria that take up unaltered plasmids are resistant to both antibiotics.

· Bacteria that take up unincorporated foreign DNA (or no DNA at all) are sensitive to both antibiotics.

· Bacteria that take up recombinant plasmids are resistant to ampicillin but sensitive to tetracycline.

· The resulting bacteria are grown on a medium that contains ampicillin.

· The survivors might have a plasmid that either contains or does not contain an insert.

· To determine the presence of the insert, the colonies on the plate containing ampicillin are copied over to a plate with tetracycline.

· Those that fail to grow are the ones likely to have the insert. This is called screening.

· The colonies that failed to grow on the tetracycline plate are selected from the ampicillin plate.

· Other methods have since been developed for screening.

· Reporter genes such as luciferase have been used. Luciferase is the enzyme that makes fireflies glow in the dark when supplied with its substrate.

· Green fluorescent protein, which is the product of a jellyfish gene, glows without any required substrate.

· Cells with this gene in the plasmid grow on ampicillin and glow in the dark. (See Figure 17.7.)

· Many vectors in common use have just a single antibiotic resistance gene outside of the sites for foreign DNA insertion.

Sources of Genes for Cloning

· DNA for insertion can be random fragments of the DNA from an organism (a DNA library).

· DNA can be generated by reverse transcription from mRNA. This DNA is called cDNA (complementary DNA).

· Organic chemists can synthesize DNA in the laboratory.

Gene libraries contain pieces of a genome

· The 23 pairs of human chromosomes can be thought of as a library that contains the entire genome of our species.

· The average size of each chromosome, or “volume,” is 80 million base pairs. Each chromosome encodes several thousand genes.

· To study them, chromosomes are sorted and fragmented. (See Figure 17.8.)

· Using plasmids for insertion of DNA, about one million separate fragments are required for the human genome library.

· Phage lambda, which carries four times as much DNA as a plasmid, is used to hold these random fragments.

· It takes about 250,000 different phages to ensure a copy of every sequence.

· This still seems like a large number, but just one growth plate can hold as many as 80,000 phage colonies.

A DNA copy of mRNA can be made

· A smaller DNA library (genes for a particular tissue) can be made from cDNA. (See Figure 17.9.)

· The poly A tail, found on many mRNA molecules from eukaryotes, makes it possible to make DNA from it.

· Reverse transcriptase of mRNA and oligo dT primer is added.

· Reverse transcriptase is an enzyme that uses an RNA template to synthesize a DNA–RNA hybrid.

· The DNA is complementary to the RNA and is called cDNA.

· cDNA's from certain cell types have been useful in discovering the nature of differential gene expression.

· Their use has shown that up to one-third of all genes of an animal are expressed only during prenatal development.

· They are useful for giving a picture of genes expressed at flow levels in only a few cell types.

DNA can be synthesized chemically in the laboratory

· If the amino acid sequence of a protein is known, it is possible to synthesize a DNA that can code for protein using organic chemistry.

· Using the knowledge of the genetic code and known amino acid sequences, the most likely base sequence for the gene may be found.

· Often sequences are added to this sequence to promote expression of the protein.

· These noncoding sequences must be the ones actually recognized by the host cell if the synthetic gene is to be transcribed.

· Human insulin has been manufactured using this approach.

DNA can be mutated in the laboratory

· Recombinant DNA technology is an effective tool for studying mutations without having to look for them in nature.

· With synthetic DNA, mutational effects can be studied by creating specific mutations.

· Additions, deletions, and base-pair substitutions can be manipulated and tracked.

· The functional importance of certain amino acid sequences can be studied.

· The signals that mark proteins for passage through the ER membrane were discovered by site-directed mutagenesis.

· When the signal sequences were removed, proteins weren’t transported across the ER membrane.

· When the signal sequences were added to a protein that normally doesn't get transported across the ER membrane, it was transported.

· Mutagenesis techniques have been successful in the design of specific drugs.

· Site-directed mutagenesis is being used to devise rules for tertiary structure of proteins.

· Site-specific mutagenesis is also being used to study the effects of the tertiary structure on enzyme activation.

Some Additional Tools for DNA Manipulation

Genes can be inactivated by homologous recombination

· Homologous recombination can be used to selectively inactivate genes.

· This manipulation is called a knockout experiment. (See Figure 17.10.)

· A gene of interest is cloned into a plasmid.

· Additional DNA is added within the gene to disable it.

· Mouse embryonic cells are transfected with the DNA.

· Homologous recombination knocks out the normal functioning copy.

· Transfected cells are identified by selecting or screening for a genetic marker included in the insert.

· These transfected cells are included into an early mouse embryo. Some of the cells end up as germ cells, and homozygous mice with a knocked-out gene can be generated.

· This technique is important to assess the roles of genes during development.

DNA chips can reveal DNA mutations and RNA expression

· There are a large number of genes in eukaryotic genomes.

· The pattern of expression between different tissues and at different times is quite distinctive.

· Cells have unique mRNA's.

· For example, early-stage skin cancer cells have a unique mRNA "fingerprint."

· To find these patterns, DNA sequences have to be arranged in an array on some solid support.

· DNA chip technology provides these large arrays of sequences for hybridization. (See Figure 17.11.)

· Merging DNA technology with the manufacturing technology of the semiconductor industry, large arrays are being produced.

· DNA chips are glass slides onto which DNA sequences are attached in precise order.

· The typical slide is divided into 24 ´ 24 µm squares.

· Each contains about 10 million copies of a particular sequence, which is up to 20 nucleotides long.

· A computer controls the additions of the nucleotides in a predetermined pattern.

· Up to 60,000 different sequences can be put on a single chip.

· Cellular mRNA is isolated from cells and when incubated with reverse transcriptase (RT) is used to make complementary DNA (cDNA).

· The cDNA is amplified by PCR prior to hybridization.

· Reverse transcriptase and PCR are used together in a process called RT-PCR.

· The amplified cDNA is coupled to a fluorescent dye. It is then hybridized to the chip.

· A sensitive scanner detects the spots on the array that glow. The combinations of spots that light up differ with different types of cells or different physiological states.

· DNA chip technology can be used in detecting genetic variants.

· It can be difficult to sequence an entire gene to look for mutations.

· It is possible to make a chip with 20-nucleotide fragments along the gene in every possible mutant sequence.

· Probing a person's DNA determines whether any of the DNA hybridized to a mutant sequence on the chip.

· This method may provide a quick way to detect human mutations.

Antisense RNA and ribozymes can prevent the expression of specific genes

· Base-pairing rules can be used to make genes and also to stop mRNA translation.

· Antisense RNA is RNA that is complementary to a sequence of mRNA. (See Figure 17.12.)

· The antisense RNA forms a double-stranded RNA hybrid with an mRNA molecule, preventing tRNA from binding to that mRNA.

· These hybrids are broken down rapidly in the cytoplasm, so although the gene is transcribed, translation does not occur.

· In the laboratory, either antisense RNA or DNA that codes for it is introduced into cells.

· A tissue-specific promoter makes it possible to have mRNA inactivation occur only in a certain type of cell so that expression only occurs in targeted tissue.

· Some antisense RNA has been coupled with another RNA sequence, a ribozyme, which destroys the target mRNA by cleavage.

· Antisense RNA technology has been used to test cause-and-effect relationships.

· It has provided volumes of information on development, cancer, and other important biological questions.

Biotechnology: Applications of DNA Manipulation

· Biotechnology is the use of microbial, plant, and animal cells to produce materials useful to people.

· These products include foods, medicines, and chemicals.

· Beer and wine are examples of biotechnology that has been around for at least 8000 years.

· The use of bacterial cultures to make cheese and yogurt has gone on for centuries.

· The works of Pasteur and Fleming are classic examples of using microbes to make certain products.

· Modern molecular biology has vastly increased the number of products beyond those that are naturally made by microbes.

· Gene cloning has made this possible.

Expression vectors can turn cells into protein factories

· Expression vectors are typical vectors, but they also have bacterial promoters, which are needed in the transcription of the foreign DNA. (See Figure 17.13.)

· An expression vector might have an inducible promoter, which can be stimulated into expression by responding to a specific signal such as a hormone.

· A tissue-specific promoter is expressed just in a certain tissue at a certain time.

· Targeting sequences are sometimes added to direct the protein product to an appropriate destination.

Medically useful proteins can be made by DNA technology

· Many medical products have been made using recombinant DNA technology. Hundreds more are in various stages of development. (See Table 17.1.)

· Tissue plasminogen activator:

· TPA is produced by cells lining the blood vessels.

· This enzyme converts plasminogen, found normally in the blood, into plasmin, a protein that dissolves clots. (See Figure 17.14.)

· Plasminogen activator is currently being produced in E. coli by recombinant DNA techniques.

· This new drug has been useful in treating patients who have suffered heart attacks or strokes.

· The only drug available before TPA was streptokinase, which is a bacterial enzyme and causes immune reactions if used repeatedly on a patient.

· Erythropoietin:

· This protein hormone stimulates the production of red blood cells and is produced by the kidneys.

· Those with kidney disease often fail to produce enough erythropoietin.

· Currently, transgenic bacteria produce large amounts of the protein.

· Human insulin:

· People with certain forms of diabetes mellitus have a deficiency of pancreatic insulin.

· In the past, insulin was obtained from pig pancreases. This required difficult purification procedures and caused risks of infectious disease and allergic reactions.

· Ideally, human insulin is the best, but nearly impossible to get.

· Human insulin is now made in bacteria by inserting two genes via an expression vector.

· This was the first human protein to be made for drug use.

DNA manipulation is changing agriculture

· Selective breeding has been used for centuries to improve plant and animal species to meet human needs.

· Desired phenotypes existing in nature were selected and bred, leading to improved offspring.

· Molecular biology is accelerating progress in these applications. (See Table 17.2.)

· Two major advantages over traditional techniques are that specific genes can be affected and genes can be introduced from other organisms.

· Regeneration of whole plants by cloning has made the process much faster.

· Nutritional properties have been improved.

· Edible crops have been modified to make oral vaccines.

· Plants that make their own insecticides:

· Insecticides tend to be nonspecific, killing both pest and beneficial insects.

· They are applied to the surface of plants and tend to be blown or washed away to contaminate and pollute unintended areas.

· Bacillus thuringiensis bacteria produce a protein toxin that kills the insect larvae that prey on them.

· The toxicity is 80,000 times that of the typical chemical insecticide.

· Transgenic tomato, corn, potato, and cotton plants have been made that produce a toxin from B. thuringiensis. They show considerable resistance to insects that normally feed on them.

· Cloned animals that express useful genes:

· Dolly, the cloned sheep, is transgenic and produces human a-1-antitrypsin in her milk. (See Figure 17.15.)

· This protein in humans inhibits elastase, which hydrolyzes connective tissue.

· Excess elastase is found on the surface of the lungs of people with cystic fibrosis.

· It is partially responsible for their severe breathing problems.

· The promoter for lactoglobulin, a protein secreted into milk, was attached to the gene for a-1AT, and this was introduced into a fertilized sheep egg.

· The process of producing pharmaceuticals using agriculture is nicknamed "pharming."

· Goats, sheep, and cows are all being used for the production of medically useful products in milk.

· These products include blood clotting factors and antibodies for treating colon cancer.

· Crops that are resistant to herbicides:

· Glyphosate, trademarked Roundup, is a broad-spectrum herbicide.

· It kills plants by inhibiting an enzyme system in chloroplasts that is involved in the synthesis of amino acids.

· A bacterial gene is able to break down glyphosate.

· This gene, when inserted into useful crop plants, confers resistance to glyphosate.

· Fully half of certain U.S. crops (corn, cotton, soybeans) are now transgenic.

· Grains with improved nutritional characteristics:

· Rice has no b-carotene, a molecule that is converted to vitamin A in animals.

· Genes from bacteria and daffodil plants were transferred to rice using the vector Agrobacterium tumefaciens.

· Now a genetically modified strain of rice produces b-carotene. (See Figure 17.16.)

There is public concern about biotechnology

· E. coli, the bacterium used most in biotechnology, normally lives in the human intestine.

· Early concerns about biotechnology focused on the danger that genetically modified E. coli might share their genes with bacteria living in humans.

· Researchers now take precautions against this.

· For example, the strains of E. coli used in the lab have a number of mutations that make their survival in the human intestine impossible.

· As biotechnology developed, it was determined that safety fears were exaggerated.

· Since then, medical products made by DNA technology have become widely accepted.

· There is currently resistance to the introduction of genetically modified crops. The concerns generally fall into three categories:

· Genetic manipulation is an unnatural interference with nature.

· Genetically altered foods are unsafe to eat.

· Genetically altered plants are dangerous to the environment; transgenes may “escape” to other species.

· Advocates of biotechnology agree that genetic manipulation is unnatural but point out that traditional methods of genetic manipulation (hybridization and artificial selection) have already produced crops that are far removed from their natural ancestors.

· Regarding safety for human consumption, advocates of genetic engineering note that typically only single genes specific for plant function are added.

· As plant biotechnology moves from adding genes to improve plant growth to adding genes that affect human nutrition, such concerns will become more pressing.

· The risks to the environment are more difficult to assess.

· Transgenic plants undergo extensive field testing before they are approved for use, but the complexity of the biological world makes it impossible to predict all potential environmental effects of transgenic organisms.

· Because of the potential benefits of agricultural biotechnology, most scientists believe we should proceed, but with caution.

DNA fingerprinting uses the polymerase chain reaction.

· With the exception of identical twins, each human being is genetically distinct from all other human beings.

· To develop a test that can find distinctions, scientists look for DNA sequences that are highly polymorphic (genes having multiple alleles in the human population).

· Sequences called VNTRs (variable number of tandem repeats) are easily detectable if they are between two restriction enzyme recognition sites.

· Different individuals have different numbers of repeats. Each gets two sequences of repeats, one from the mother and one from the father. (See Figure 17.17.)

· Using PCR and gel electrophoresis, patterns for each individual can be determined.

· DNA from a single cell is sufficient to determine the DNA fingerprint because PCR can amplify a tiny amount of DNA in a few hours.

· DNA fingerprints have been used in forensics (murder, rape, etc.) and in contested paternity.

· The technique is more often used to establish innocence rather than guilt because the same genome sample may turn up in people with the same patterns.

· Russian Tsar family members have been studied to show that bodies recently discovered are probably related to several living descendants of the Tsar.

· Genetic testing has revealed a strong likelihood that Thomas Jefferson had an illegitimate child by a female slave, Sally Hemmings.

· This was determined using chromosome markers from Hemmings’s descendants and Jefferson’s uncle.

· California condors, which are extinct in the wild, are tested to reduce inbreeding and increase genetic variation in captive breeding populations.

· PCR is used in diagnosing infections in which the infectious agent is present in small amounts.

· Genetic diseases such as sickle-cell anemia are now diagnosable before they manifest themselves.

· New treatments are being developed based on genetic knowledge.

The Instructor’s Resource CD-ROM includes a photograph of an electrophoresis gel and a photograph of the gall produced by Agrobacterium tumefaciens.