Figure 10.10
Dolly the sheep and her first-born lamb Bonnie. Dolly was the first large mammal to be cloned. This picture shows that a cloned animal can perform many, if not all, of the same functions as a non-cloned animal.
In animal cloning, the nucleus from a somatic cell is inserted into an egg cell in which the nucleus has been removed. The resulting cell is cultivated and after a few divisions, the egg cell is placed into a uterus where it is allowed to develop into a fetus that is genetically identical to the donor of the original nucleus (Figure below). For an animation of cloning, see http://www.dnalc.org/resources/animations/cloning101.html.
Figure 10.11
Reproductive cloning: The nucleus is removed from a somatic cell and fused with a denucleated egg cell. The resulting cell may develop into a colony of cloned cells, which is placed into a surrogate mother. In therapeutic cloning, the resulting cells are grown in tissue culture; an animal is not produced, but genetically identical cells are produced.
Applications of DNA Technology: Forensic DNA Analysis
You know that DNA can be used to distinguish individuals from each other. You may have heard that DNA can also be used to match evidence and suspects and help solve crimes. This is demonstrated on shows like CSI: Crime Scene Investigation. But how is this done? How is a “genetic fingerprint,” a DNA pattern unique to each individual (except identical twins) created? Genetic fingerprinting, or DNA fingerprinting, distinguishes between individuals of the same species using only samples of their DNA. DNA fingerprinting has thus become one of the most powerful tools of the forensic scientist, enabling law enforcement personnel to match biological evidence from crime scenes to suspects. As any two humans have the majority of their DNA sequence in common, those sequences which demonstrate high variability must be analyzed. This DNA analysis was first developed using DNA hybridization techniques, but now is almost exclusively PCR-based.
DNA fingerprinting was developed by Sir Alec Jeffreys in 1985. Genetic fingerprinting exploits highly variable repeating sequences. Two categories of these sequences are microsatellites and minisatellites. Microsatellites, also known as short tandem repeats (STRs), consist of adjacent repeating units of 2 - 10 bases in length, for example (GATC)n, where GATC is a tetranucleotide (4 base) repeat and n refers to the number of repeats. It is the number of repeating units at a given locus that is variable. An STR profile can be created for any individual by analyzing a series of STRs (Figure below). Two unrelated humans will be unlikely to have the same numbers of repeats at a given locus.
In STR profiling, PCR is used to obtain enough DNA to then detect the number of repeats at 13 specific loci. PCR products are separated by gel or capillary electrophoresis. By examining enough STR loci and counting how many repeats of a specific STR sequence there are at a given locus, it is possible to create a unique genetic profile of an individual. STR analysis has become the prevalent analysis method for determining genetic profiles in forensic cases. It is possible to establish a match that is extremely unlikely to have arisen by coincidence, except in the case of identical twins, who will have identical genetic profiles. The polymorphisms (different in the number of repeats) displayed at each STR region will be shared by approximately 5 - 20% of individuals. When analyzing STRs at multiple loci, such as the 13 STRs analyzed in forensic DNA analysis, it is the unique combinations of these polymorphisms in an individual that makes this method unmatched as an identification tool. The more STR regions that are analyzed in an individual the more discriminating the test becomes.
Capillary electrophoresis is similar to gel electrophoresis but uses a capillary tube filled with the gelatin material.
Genetic fingerprinting is used in forensic science to match suspects to samples of blood, hair, saliva or semen, or other sources of DNA. It has also led to several exonerations of formerly convicted suspects. Genetic fingerprinting is also used for identifying human remains, testing for paternity, matching organ donors, studying populations of wild animals, and establishing the province or composition of foods. It has also been used to generate hypotheses on the pattern of the human migration.
In the United States, DNA fingerprint profiles generated from the 13 STR loci are stored in CODIS, The Combined DNA Index System, maintained by the Federal Bureau of Investigation. As of 2007, CODIS maintained over 4.5 million profiles. Profiles maintained in CODIS are compiled from both suspects and evidence, and therefore are used to help solve criminal cases. Profiles of missing persons are also maintained in CODIS. The true power of STR analysis is in its statistical power of discrimination. Because the 13 loci are independently assorted, the laws of probabilities can be applied. This means that if someone has the genotype of ABC at three independent loci, then the probability of having that specific genotype is the probability of having type A times the probability of having type B times the probability of having type C. This has resulted in the ability to generate match probabilities of 1 in a quintillion (1 with 18 zeros after it) or more, that is, the chance of two samples matching by coincidence is greater than the number of people on the planet, or the number of people that have ever lived!
Figure 10.12
The CODIS loci analyzed by STR analysis. Notice they are spread over 14 chromosomes, and that two are on the X and Y chromosomes.
The development of PCR has enabled STR analysis to become the method of choice for DNA identification. Prior to PCR, other methods were utilized. These include restriction fragment length polymorphism (RFLP) analysis and Southern blot analysis.
RFLP Analysis: Restriction Fragment Length Polymorphism
Prior to the development of PCR, restriction enzyme digestion of DNA followed by Southern blot analysis was used for DNA fingerprinting. This analysis is based on the polymorphic nature of restriction enzyme sites among different individuals, hence restriction fragment length polymorphisms are formed after digestion of DNA with these enzymes. A Southern blot, named after its inventor Edwin Southern, is a method used to check for the presence of a specific DNA sequence in a DNA sample. Once an individual’s DNA is digested with a specific restriction enzyme, the resulting fragments are analyzed by Southern blot analysis. These fragments will produce a specific pattern for that individual. Southern blotting is also used for other molecular biology procedures, including gene identification and isolation. Other blotting methods that employ similar principles have been developed. These include the western blot and northern blot. These procedures analyze proteins and RNA respectively.
RFLP and Southern blot analysis involved several steps:
First, the DNA being analyzed is cut into different-sized pieces using restriction enzymes.
The resulting DNA fragments are separated by gel electrophoresis.
Next, an alkaline solution or heat is applied to the gel so that the DNA denatures and separates into single strands.
Nitrocellulose paper is pressed evenly against the gel and then baked so the DNA is permanently attached to it. The DNA is now ready to be analyzed using a radioactive single-stranded DNA probe in a hybridization reaction.
After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on X-ray film by autoradiography (Figure below).
Figure 10.13
Mutations can create or abolish restriction enzyme (RE) recognition sites, thus affecting quantities and length of DNA fragments resulting from RE digestion.
Hybridization is when two genetic sequences bind together because of the hydrogen bonds that form between the base pairs. To make hybridization work, the radioactive probe has to be denatured so that it is single-stranded. The denatured probe and the Southern blot are incubated together, allowing the probe to bind to the corresponding fragment on the Southern blot. The probe will bond to the denatured DNA wherever it finds a fit. Hybridization of a probe made to a variable segment of DNA will produce a DNA fingerprint pattern specific for an individual. This procedure has a number of steps and is very labor intensive.
PCR-based methods are much simpler.
Ethical, Legal, and Social Issues
Imagine someone analyzes part of your DNA. Who controls that information? What if your health insurance company found out you were predisposed to develop a devastating genetic disease. Might they decide to cancel your insurance?
Privacy issues concerning genetic information is a growing issue in this day and age, especially among those who donate DNA for large-scale sequence-variation studies. Other concerns have been to anticipate how the resulting data may affect concepts of race and ethnicity; identify potential uses (or misuses) of genetic data in workplaces, schools, and courts; identify commercial uses; and foresee impacts of genetic advances on the concepts of humanity and personal responsibility.
ELSI stands for Ethical, Legal and Social Issues. It's a term associated with the Human Genome project. This project didn't only have the goal to identify all the approximately 20,000 – 24,000 genes in the human genome, but also to address the ELSI that might arise from the project. The U.S. Department of Energy (DOE) and the National Human Genome Research Institute (NHGRI) of the National Institutes of Health (NIH) devoted 3% to 5% of their annual human genome research budget toward studying ethical, legal, and social issues surrounding the availability of your genetic information. This represents the world's largest bioethics program and has become a model for ELSI programs around the world.
Rapid advances in DNA-based research, human genetics, and their applications have resulted in new and complex ethical and legal issues for society. ELSI programs that identify and address these implications have been an integral part of the Human Genome Project since its inception. These programs have resulted in a body of work that promotes education and helps guide the conduct of genetic research and the development of related medical and public policies.
ELSI programs address the following issues, among others:
Privacy and confidentiality issues concerning personal genetic information.
The fairness in the use of personal genetic information by insurers, employers, courts, schools, adoption agencies, and the military, among others.
The psychological impact and stigmatization due to an individual's genetic differences.
Clinical issues. These include the education of doctors and other health service providers, patients, and the general public in the capabilities and uses of genetic information, and the scientific/medical limitations of genetic testing. Clinical issues also include the implementation of standards and quality-control measures in genetic testing procedures.
Reproductive issues. These include adequate informed consent for complex and potentially controversial procedures, and the use of genetic information in making decisions concerning reproductive options.
Uncertainties associated with genetic testing. The current and future uncertainties associated with testing for susceptibilities to a genetic condition raise many ethical issues, as does the testing for predisposition to a complex condition (such as heart disease) linked to multiple genes and gene-environment interactions.
Health and environmental issues concerning genetically modified foods and microbes.
Commercialization of genetic products including property rights, such as patents and copyrights, and issues concerning the accessibility to genetic data and materials.
Biotechnology will have a tremendous impact on our future - of this there is no doubt. Is society entering some dangerous areas? Well, many of these issues have never been analyzed until now. With the discovery of countless amounts of genetic information and the development of its applications, many questions need to be addressed.
Who should have access to personal genetic information, and how will it be used?
Who owns and controls genetic information?
How does personal genetic information affect an individual and society's perceptions of that individual?
How does genomic information affect members of minority communities?
How reliable and useful is fetal genetic testing?
How will genetic tests be evaluated and regulated for accuracy, reliability, and utility?
How do we prepare the public to make informed choices?
Should testing be performed when no treatment is available?
Should parents have the right to have their minor children tested for adult-onset diseases?
Are genetic tests reliable and interpretable by the medical community?
Where is the line between medical treatment and enhancement?
Are genetically modified foods and other products safe for humans and the environment?
How will these technologies affect developing nations' dependence on the West?
Who owns genes and other pieces of DNA?
Will patenting DNA sequences limit their accessibility and development into useful products?
Are scientific fantasies, such as those depicted on TV shows such as Star Trek or in the movie GATTACA, a possibility? Who can really say? How, really, will biotechnology affect our future? It seems as if the possibilities are endless.
Lesson Summary
In medicine, modern biotechnology provides significant applications in such areas as pharmacogenomics, genetic testing (prenatal diagnosis), and gene therapy.
Pharmacogenomics, the combination of pharmacology and genomics, is the study of the relationship between pharmaceuticals and genetics.
Pharmacogenomics will result in the following benefits:
Development of tailor-made medicines.
More accurate methods of determining appropriate drug dosages.
Improvements in the drug discovery and approval process.
Better vaccines.
Genetic testing involves the direct examination of DNA sequences.
Genetic testing can be used to: diagnose a disease; confirm a diagnosis; provide prognostic information about the course of a disease; confirm the existence of a disease; predict the risk of future development of a disease in otherwise healthy individuals or their children; screen for carriers (unaffected individuals who are heterozygous for a disease gene); perform prenatal diagnostic screening; and perform newborn screening.
Biotechnology in agriculture includes the development of transgenic crops - the placement of genes into plants to give the crop a beneficial trait. Benefits include improved yield from crops, reduced vulnerability of crops to environmental stresses, increased nutritional qualities of food crops, improved taste, texture or appearance of food, reduced dependence on fertilizers, pesticides and other agrochemicals, and production of vaccines.
Transgenic animals are animals that have incorporated a gene from another species into their genome. They are used as experimental models to perform phenotypic tests with genes whose function is unknown, or to generate animals that are susceptible to certain compounds or stresses for testing purposes. Other applications include the production of human hormones, such as insulin.
Animal cloning is the generation of genetically identical animals using DNA from a donor animal, not a gamete. Dolly, a sheep, was the first mammal to be cloned from an adult somatic cell.
Genetic fingerprinting, or DNA fingerprinting, distinguishes between individuals of the same species using only samples of their DNA. DNA fingerprinting has thus become one of the most powerful tools of the forensic scientist, enabling law enforcement personnel to match biological evidence from crime scenes to suspects.
ELSI stands for Ethical, Legal and Social Issues. This is a term associated with the Human Genome project. Rapid advances in DNA-based research, human genetics, and their applications have resulted in new and complex ethical and legal issues for society. ELSI programs that identify and address these implications have been an integral part of the Human Genome Project since its inception. These programs have resulted in a body of work that promotes education and helps guide the conduct of genetic research and the development of related medical and public policies.
Review Questions
List applications of DNA
technology.
List how DNA technology is used in agriculture.
How is DNA technology used in medicine?
What are some of the benefits of pharmacogenomics?
Describe how pharmacogenomics will result in specialty medicines.
What are potential uses of genetic testing?
Describe how DNA technology can improve yield from crops.
Describe how DNA technology can be used to reduce vulnerability to environmental stresses. Why is this important? State an example.
What is the difference between a transgenic animal and a cloned animal?
Who was Dolly? Why was she important?
What is a DNA fingerprint and how is it used?
What is STR profiling?
Describe why ELSI programs are important.
List some ELSI issues.
Further Reading / Supplemental Links
http://www.genome.gov
http://www.dna.gov/basics/http://www.ornl.gov/sci/techresources/Human_Genome/medicine/pharma.shtml
http://www.ornl.gov/sci/techresources/Human_Genome/medicine/pharma.shtml
http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml
CK-12 Biology I - Honors Page 44
