17: Biotechnology and Genomics Flashcards
Biotechnology, Mapping Genomes, Whole-Genome Sequencing, Applying Genomics, Genomics and Proteomics (161 cards)
1
Q
What is antibiotic resistance?
A
The ability of an organism to be unaffected by the actions of an antibiotic.
2
Q
What is biotechnology?
A
The use of biological agents for technological advancement.
3
Q
What is cellular cloning?
A
The production of identical cell populations by binary fission.
4
Q
What is a clone?
A
An exact replica.
5
Q
What is foreign DNA?
A
DNA that belongs to a different species or DNA that is artificially synthesized. AKA transgene.
6
Q
What is gel electrophoresis?
A
A technique used to separate molecules on the basis of size using electric charge.
7
Q
What is gene targeting?
A
A method for altering the sequence of a specific gene by introducing the modified version on a vector.
8
Q
What is gene therapy?
A
A technique used to cure inheritable diseases by replacing mutant genes with good genes.
9
Q
What is genetic diagnosis?
A
Diagnosis of the potential for disease development by analyzing disease-causing genes.
10
Q
What is genetic engineering?
A
Alteration of the genetic makeup of an organism.
11
Q
What is genetic testing?
A
The process of testing for the presence of disease-causing genes.
12
Q
What is a genetically modified organism (GMO)?
A
An organism whose genome has been artificially changed.
13
Q
What is host DNA?
A
DNA that is present in the genome of the organism of interest.
14
Q
What is a lysis buffer?
A
A solution used to break the cell membrane and release cell contents.
15
Q
What is molecular cloning?
A
Cloning of DNA fragments.
16
Q
What is a multiple cloning site (MCS)?
A
A site that can be recognized by multiple restriction endonucleases.
17
Q
What is northern blotting?
A
The transfer of RNA from a gel to a nylon membrane.
18
Q
What is polymerase chain reaction (PCR)?
A
A technique used to amplify DNA.
19
Q
What is a probe?
A
A small DNA fragment used to determine if the complementary sequence is present in a DNA sample.
20
Q
What is a protease?
A
An enzyme that breaks down proteins.
21
Q
What is recombinant DNA?
A
Combination of DNA fragments generated by molecular cloning that does not exist in nature.
22
Q
What is a recombinant protein?
A
A protein product of a gene derived by molecular cloning (recombinant DNA).
23
Q
What is reproductive cloning?
A
Cloning of entire organisms.
24
Q
What is a restriction endonuclease?
A
An enzyme that can recognize and cleave specific DNA sequences.
25
What are reverse genetics?
A method of determining the function of a gene by starting with the gene itself instead of starting with the gene product.
26
What is reverse transcriptase PCR (RT-PCR)?
A PCR technique that involves converting RNA to DNA (cDNA) by reverse transcriptase.
27
What is a ribonuclease?
An enzyme that breaks down RNA.
28
What is southern blotting?
The transfer of DNA from a gel to a nylon membrane.
29
What is a Ti plasmid?
A plasmid system derived from *Agrobacterium tumifaciens* that has been used by scientists to introduce foreign DNA into plant cells.
30
What is a transgenic organism?
An organism that receives DNA from a different species.
31
What are some examples of the uses of biotechnology?
The primary applications of this technology are in medicine (production of vaccines and antibiotics) and agriculture (genetic modification of crops, such as to increase yields). Biotechnology also has many industrial applications, such as fermentation, the treatment of oil spills, and the production of biofuels.
32
How is DNA extracted?
Various techniques are used to extract different types of DNA. Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired (such as degradation of unwanted molecules and separation from the DNA sample). Cells are broken using a lysis buffer. These enzymes break apart lipid molecules in the cell membranes and nuclear membranes. Macromolecules are inactivated using enzymes such as proteases that break down proteins, and ribonucleases (RNAses) that break down RNA. The DNA is then precipitated using alcohol. Human genomic DNA is usually visible as a gelatinous, white mass. The DNA samples can be stored frozen at –80°C for several years.
33
Why is RNA analysis performed?
RNA analysis is performed to study gene expression patterns in cells.
34
How is RNA extracted?
RNA is naturally very unstable because RNAses are commonly present in nature and very difficult to inactivate. Similar to DNA, RNA extraction involves the use of various buffers and enzymes to inactivate macromolecules and preserve the RNA.
35
How does gel electrophoresis work?
Because nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, they can be mobilized by an electric field. Gel electrophoresis is a technique used to separate molecules on the basis of size, using this charge. The nucleic acids can be separated as whole chromosomes or fragments. The nucleic acids are loaded into a slot near the negative electrode of a semisolid, porous gel matrix and pulled toward the positive electrode at the opposite end of the gel. Smaller molecules move through the pores in the gel faster than larger molecules; this difference in the rate of migration separates the fragments on the basis of size. There are molecular weight standard samples that can be run alongside the molecules to provide a size comparison. Nucleic acids in a gel matrix can be observed using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the top of the gel (the negative electrode end) on the basis of their size. A mixture of genomic DNA fragments of varying sizes appear as a long smear, whereas uncut genomic DNA is usually too large to run through the gel and forms a single large band at the top of the gel.
36
What is PCR used for?
Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA for further analysis. PCR is used for many purposes in laboratories, such as the cloning of gene fragments to analyze genetic diseases, identification of contaminant foreign DNA in a sample, and the amplification of DNA for sequencing. More practical applications include the determination of paternity and detection of genetic diseases.
37
How does PCR work?
Polymerase chain reaction, or PCR, is used to amplify a specific sequence of DNA. Primers—short pieces of DNA complementary to each end of the target sequence—are combined with genomic DNA, Taq polymerase, and deoxynucleotides. Taq polymerase is a DNA polymerase isolated from the thermostable bacterium *Thermus aquaticus* that is able to withstand the high temperatures used in PCR. *Thermus aquaticus* grows in the Lower Geyser Basin of Yellowstone National Park.
The PCR cycle consists of three steps–denaturation, annealing, and DNA synthesis–that occur at high, low, and intermediate temperatures, respectively. The cycle is repeated again and again, resulting in a doubling of DNA molecules each time. After several cycles, the vast majority of strands produced are the same length as the distance between the two primers.
In step 1, denaturation, the sample is heated to a high temperature so the DNA strands separate.
In step 2, annealing, the sample is cooled so the primer can anneal to the DNA.
In step 3, DNA synthesis, the sample is warmed. Taq polymerase synthesizes new strands of DNA.
38
How does reverse transcriptase PCR work?
DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR). The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it.
39
How does northern and southern blotting work?
Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences. Short DNA fragments called probes are designed and labeled with radioactive or fluorescent dyes to aid detection. Gel electrophoresis separates the nucleic acid fragments according to their size. The fragments in the gel are then transferred onto a nylon membrane in a procedure called blotting. The nucleic acid fragments that are bound to the surface of the membrane can then be probed with specific radioactively or fluorescently labeled probe sequences.
40
What is northern and southern blotting used for?
Southern blots are used to detect the presence of certain DNA sequences in a given genome, and northern blots are used to detect gene expression.
41
What is western blotting?
The transfer of protein from a gel to a nylon membrane and detected using antibodies.
42
What is the difference between reproductive and molecular cloning?
The re-creation of a whole organism is referred to as “reproductive cloning.” The reproduction of regions or fragments of the genome is referred to as molecular cloning.
43
What is molecular cloning used for?
Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products), or noncoding regions in isolation.
44
What is a plasmid?
A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA.
Plasmids occur naturally in bacterial populations (such as *Escherichia coli*) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics).
45
How are plasmids used in molecular cloning?
In cloning, the plasmid molecules can be used to provide a "folder" in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of DNA from the human genome (or the genome of another organism that is being studied) is referred to as foreign DNA, or a transgene, to differentiate it from the DNA of the bacterium, which is called the host DNA.
Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS).
46
How do restriction endonucleases work?
Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease.
47
What is another term for recombinant DNA?
Recombinant DNA molecules are also called chimeric molecules because the origin of different parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis.
48
When will genes not be expressed using recombinant DNA?
Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to be moved into a different vector (or host) that is better designed for gene expression. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins.
49
What is parthenogenesis?
Parthenogenesis, or “virgin birth,” occurs when an embryo grows and develops without the fertilization of the egg occurring; this is a form of asexual reproduction.
50
What are some examples of parthenogenesis in nature?
An example of parthenogenesis occurs in species in which the female lays an egg and if the egg is fertilized, it is a diploid egg and the individual develops into a female; if the egg is not fertilized, it remains a haploid egg and develops into a male. The unfertilized egg is called a parthenogenic, or virgin, egg. Some insects and reptiles lay parthenogenic eggs that can develop into adults.
51
How does reproductive cloning work?
Sexual reproduction requires two cells; when the haploid egg and sperm cells fuse, a diploid zygote results. The zygote nucleus contains the genetic information to produce a new individual. However, early embryonic development requires the cytoplasmic material contained in the egg cell. This idea forms the basis for reproductive cloning. Therefore, if the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of any individual of the same species (called a donor), it will become a zygote that is genetically identical to the donor.
52
What is a technique for reproductive cloning?
Somatic cell nuclear transfer is the technique of transferring a diploid nucleus into an enucleated egg. It can be used for either therapeutic cloning or reproductive cloning.
53
How has reproductive cloning been used in animals?
The first cloned animal was Dolly, a sheep who was born in 1996. The success rate of reproductive cloning at the time was very low. Dolly lived for seven years and died of respiratory complications. There is speculation that because the cell DNA belongs to an older individual, the age of the DNA may affect the life expectancy of a cloned individual. Since Dolly, several animals such as horses, bulls, and goats have been successfully cloned, although these individuals often exhibit facial, limb, and cardiac abnormalities.
54
How has reproductive cloning been used in humans?
There have been attempts at producing cloned human embryos as sources of embryonic stem cells, sometimes referred to as cloning for therapeutic purposes. Therapeutic cloning produces stem cells to attempt to remedy detrimental diseases or defects (unlike reproductive cloning, which aims to reproduce an organism). Still, therapeutic cloning efforts have met with resistance because of bioethical considerations.
55
How is genetic engineering performed?
Genetic engineering is the alteration of an organism’s genotype using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits. The addition of foreign DNA in the form of recombinant DNA vectors generated by molecular cloning is the most common method of genetic engineering.
56
What are some examples of GMOs?
Bacteria, plants, and animals have been genetically modified since the early 1970s for academic, medical, agricultural, and industrial purposes. In the US, GMOs such as Roundup-ready soybeans and borer-resistant corn are part of many common processed foods.
57
How is gene targeting performed?
Mutating or deleting genes provides researchers with clues about gene function. The methods used to disable gene function are collectively called gene targeting. Gene targeting is the use of recombinant DNA vectors to alter the expression of a particular gene, either by introducing mutations in a gene, or by eliminating the expression of a certain gene by deleting a part or all of the gene sequence from the genome of an organism.
58
How is genetic testing used?
The process of testing for suspected genetic defects before administering treatment is called genetic diagnosis by genetic testing. Depending on the inheritance patterns of a disease-causing gene, family members are advised to undergo genetic testing. For example, women diagnosed with breast cancer are usually advised to have a biopsy so that the medical team can determine the genetic basis of cancer development. Treatment plans are based on the findings of genetic tests that determine the type of cancer. If the cancer is caused by inherited gene mutations, other female relatives are also advised to undergo genetic testing and periodic screening for breast cancer. Genetic testing is also offered for fetuses (or embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with specific debilitating diseases.
59
How is gene therapy used?
Gene therapy is a genetic engineering technique used to cure disease. In its simplest form, it involves the introduction of a good gene at a random location in the genome to aid the cure of a disease that is caused by a mutated gene. The good gene is usually introduced into diseased cells as part of a vector transmitted by a virus that can infect the host cell and deliver the foreign DNA. More advanced forms of gene therapy try to correct the mutation at the original site in the genome, such as is the case with treatment of severe combined immunodeficiency (SCID)
60
How are vaccines produced?
Traditional vaccination strategies use weakened or inactive forms of microorganisms to mount the initial immune response. Modern techniques use the genes of microorganisms cloned into vectors to mass produce the desired antigen. The antigen is then introduced into the body to stimulate the primary immune response and trigger immune memory. Genes cloned from the influenza virus have been used to combat the constantly changing strains of this virus.
61
How are antibiotics produced?
Antibiotics are a biotechnological product. They are naturally produced by microorganisms, such as fungi, to attain an advantage over bacterial populations. Antibiotics are produced on a large scale by cultivating and manipulating fungal cells.
62
What are some examples of recombinant DNA used for hormone production?
Recombinant DNA technology was used to produce large-scale quantities of human insulin in E. coli as early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in humans because of differences in the gene product. In addition, human growth hormone (HGH) is used to treat growth disorders in children. The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector.
63
How have transgenic animals been used?
Although several recombinant proteins used in medicine are successfully produced in bacteria, some proteins require a eukaryotic animal host for proper processing. For this reason, the desired genes are cloned and expressed in animals, such as sheep, goats, chickens, and mice. Animals that have been modified to express recombinant DNA are called transgenic animals. Several human proteins are expressed in the milk of transgenic sheep and goats, and some are expressed in the eggs of chickens. Mice have been used extensively for expressing and studying the effects of recombinant genes and mutations.
64
How have transgenic plants been used?
Manipulating the DNA of plants (i.e., creating GMOs) has helped to create desirable traits, such as disease resistance, herbicide and pesticide resistance, better nutritional value, and better shelf-life. Plants are the most important source of food for the human population. Farmers developed ways to select for plant varieties with desirable traits long before modern-day biotechnology practices were established. Plants that have received recombinant DNA from other species are called transgenic plants.
65
How are transgenic plants regulated?
Because they are not natural, transgenic plants and other GMOs are closely monitored by government agencies to ensure that they are fit for human consumption and do not endanger other plant and animal life. Because foreign genes can spread to other species in the environment, extensive testing is required to ensure ecological stability.
66
What are some examples of genetically-modified plants?
Staples like corn, potatoes, and tomatoes were the first crop plants to be genetically engineered.
67
What is *Agrobacterium tumefaciens*?
In plants, tumors caused by the bacterium *Agrobacterium tumefaciens* occur by transfer of DNA from the bacterium to the plant. Although the tumors do not kill the plants, they make the plants stunted and more susceptible to harsh environmental conditions. Many plants, such as walnuts, grapes, nut trees, and beets, are affected by *A. tumefaciens*.
68
How are Ti plasmids used to genetically-modify plants?
The artificial introduction of DNA into plant cells is more challenging than in animal cells because of the thick plant cell wall. Researchers used the natural transfer of DNA from *Agrobacterium* to a plant host to introduce DNA fragments of their choice into plant hosts. In nature, the disease-causing *A. tumefaciens* have a set of plasmids, called the Ti plasmids (tumor-inducing plasmids), that contain genes for the production of tumors in plants. DNA from the Ti plasmid integrates into the infected plant cell’s genome. Researchers manipulate the Ti plasmids to remove the tumor-causing genes and insert the desired DNA fragment for transfer into the plant genome. The Ti plasmids carry antibiotic resistance genes to aid selection and can be propagated in *E. coli* cells as well.
69
What is *Bacillus thuringiensis* (Bt)?
*Bacillus thuringiensis* (Bt) is a bacterium that produces protein crystals during sporulation that are toxic to many insect species that affect plants. Bt toxin has to be ingested by insects for the toxin to be activated. Insects that have eaten Bt toxin stop feeding on the plants within a few hours. After the toxin is activated in the intestines of the insects, death occurs within a couple of days.
70
How is Bt used as an insecticide?
Modern biotechnology has allowed plants to encode their own crystal Bt toxin that acts against insects. The crystal toxin genes have been cloned from Bt and introduced into plants. Bt toxin has been found to be safe for the environment, non-toxic to humans and other mammals, and is approved for use by organic farmers as a natural insecticide.
71
What was the Flavr Savr Tomato?
The first GM crop to be introduced into the market was the Flavr Savr Tomato produced in 1994. Antisense RNA technology was used to slow down the process of softening and rotting caused by fungal infections, which led to increased shelf life of the GM tomatoes. Additional genetic modification improved the flavor of this tomato. The Flavr Savr tomato did not successfully stay in the market because of problems maintaining and shipping the crop.
72
What is cytogenetic mapping?
A technique that uses a microscope to create a map from stained chromosomes.
73
What is an expressed sequence tag (EST)?
A short STS (sequence-tagged site) that is identified with cDNA.
74
What is a genetic map?
An outline of genes and their location on a chromosome.
75
What is a genetic marker?
A gene or sequence on a chromosome with a known location that is associated with a specific trait.
76
What is genetic recombination?
An exchange of DNA between homologous pairs of chromosomes.
77
What is genome mapping?
The process of finding the location of genes on each chromosome.
78
What is a cDNA library?
A collection of cloned cDNA sequences.
79
What is a genomic library?
A collection of cloned DNA which represents all of the sequences and fragments from a genome.
80
What is genomics?
The study of entire genomes including the complete set of genes, their nucleotide sequence and organization, and their interactions within a species and with other species
81
What is linkage analysis?
A procedure that analyzes the recombination of genes to determine if they are linked.
82
What is a microsatellite polymorphism?
A variation between individuals in the sequence and number of repeats of microsatellite DNA.
83
What is a physical map?
A representation of the physical distance between genes or genetic markers.
84
What is radiation hybrid mapping?
Information obtained by fragmenting the chromosome with x-rays.
85
What is a restriction fragment length polymorphism (RFLP)?
Variation between individuals in the length of DNA fragments generated by restriction endonucleases.
86
What is sequence mapping?
Mapping information obtained after DNA sequencing.
87
What is a single nucleotide polymorphism (SNP)?
Variation between individuals in a single nucleotide.
88
What is variable number of tandem repeats (VNTRs)?
Variation in the number of tandem repeats between individuals in the population.
89
What is the difference between genetic maps and physical maps?
A genetic map is an illustration that lists genes and their location on a chromosome. Genetic maps provide the big picture and use genetic markers. Physical maps present the intimate details of smaller regions of the chromosomes. A physical map is a representation of the physical distance, in nucleotides, between genes or genetic markers. Both genetic linkage maps and physical maps are required to build a complete picture of the genome.
90
What are some uses of genome mapping?
Having a complete map of the genome makes it easier for researchers to study individual genes. Human genome maps help researchers in their efforts to identify human disease-causing genes related to illnesses like cancer, heart disease, and cystic fibrosis. Genome mapping can be used in a variety of other applications, such as using live microbes to clean up pollutants or even prevent pollution. Research involving plant genome mapping may lead to producing higher crop yields or developing plants that better adapt to climate change.
91
How were the first genetic maps created?
The study of genetic maps begins with linkage analysis, a procedure that analyzes the recombination frequency between genes to determine if they are linked or show independent assortment. The term linkage was used before the discovery of DNA. Early geneticists relied on the observation of phenotypic changes to understand the genotype of an organism. Shortly after Gregor Mendel (the father of modern genetics) proposed that traits were determined by what are now known as genes, other researchers observed that different traits were often inherited together, and thereby deduced that the genes were physically linked by being located on the same chromosome. The mapping of genes relative to each other based on linkage analysis led to the development of the first genetic maps.
92
What is an example of linked genes?
In experiments performed on the garden pea, it was discovered that the color of the flower and shape of the plant’s pollen were linked traits, and therefore the genes encoding these traits were in close proximity on the same chromosome.
93
How is gene linkage determined?
Linkage analysis involves studying the recombination frequency between any two genes. The greater the distance between two genes, the higher the chance that a recombination event will occur between them, and the higher the recombination frequency between them. If the recombination frequency between two genes is less than 50 percent, they are said to be linked.
94
How are genetic markers chosen?
The generation of genetic maps requires markers. Early genetic maps were based on the use of known genes as markers. More sophisticated markers, including those based on non-coding DNA, are now used to compare the genomes of individuals in a population. Although individuals of a given species are genetically similar, they are not identical; every individual has a unique set of traits. These minor differences in the genome between individuals in a population are useful for the purposes of genetic mapping. In general, a good genetic marker is a region on the chromosome that shows variability or polymorphism (multiple forms) in the population.
95
What are some genetic markers used to generate genetic maps?
Some genetic markers used in generating genetic maps are restriction fragment length polymorphisms (RFLP), variable number of tandem repeats (VNTRs), microsatellite polymorphisms, and the single nucleotide polymorphisms (SNPs).
96
How are RFLPs used as genetic markers?
RFLPs (sometimes pronounced “rif-lips”) are detected when the DNA of an individual is cut with a restriction endonuclease that recognizes specific sequences in the DNA to generate a series of DNA fragments, which are then analyzed by gel electrophoresis. The DNA of every individual will give rise to a unique pattern of bands when cut with a particular set of restriction endonucleases; this is sometimes referred to as an individual’s DNA “fingerprint.” Certain regions of the chromosome that are subject to polymorphism will lead to the generation of the unique banding pattern.
97
How are VNTRs and microsatellite polymorphisms used as genetic markers?
VNTRs are repeated sets of nucleotides present in the non-coding regions of DNA. Non-coding, or “junk,” DNA has no known biological function; however, research shows that much of this DNA is actually transcribed. While its function is uncertain, it is certainly active, and it may be involved in the regulation of coding genes. The number of repeats may vary in individual organisms of a population. Microsatellite polymorphisms are similar to VNTRs, but the repeat unit is very small.
98
What are some limitations of genetic mapping?
Because genetic maps rely completely on the natural process of recombination, mapping is affected by natural increases or decreases in the level of recombination in any given area of the genome. Some parts of the genome are recombination hotspots, whereas others do not show a propensity for recombination. For this reason, it is important to look at mapping information developed by multiple methods.
99
What methods are used to create a physical map?
There are three methods used: cytogenetic mapping, radiation hybrid mapping, and sequence mapping.
100
How does cytogenetic mapping work?
Cytogenetic mapping uses information obtained by microscopic analysis of stained sections of the chromosome. It is possible to determine the approximate distance between genetic markers using cytogenetic mapping, but not the exact distance (number of base pairs).
101
How does radiation hybrid mapping work?
Radiation hybrid mapping uses radiation, such as x-rays, to break the DNA into fragments. The amount of radiation can be adjusted to create smaller or larger fragments. This technique overcomes the limitation of genetic mapping and is not affected by increased or decreased recombination frequency.
102
How does sequence mapping work?
Sequence mapping resulted from DNA sequencing technology that allowed for the creation of detailed physical maps with distances measured in terms of the number of base pairs. The creation of genomic libraries and complementary DNA (cDNA) libraries (collections of cloned sequences or all DNA from a genome) has sped up the process of physical mapping. A genetic site used to generate a physical map with sequencing technology (a sequence-tagged site, or STS) is a unique sequence in the genome with a known exact chromosomal location. An expressed sequence tag (EST) and a single sequence length polymorphism (SSLP) are common STSs. An EST is a short STS that is identified with cDNA libraries, while SSLPs are obtained from known genetic markers and provide a link between genetic maps and physical maps.
103
Where can information from genome maps be found?
Mapping information generated in laboratories all over the world is entered into central databases, such as GenBank at the National Center for Biotechnology Information (NCBI). Efforts are being made to make the information more easily accessible to researchers and the general public. NCBI has created a genome viewer tool to simplify the data-mining process.
104
What is the chain termination method?
A method of DNA sequencing using labeled dideoxynucleotides to terminate DNA replication; it is also called the dideoxy method or the Sanger method.
105
What is a contig?
A larger sequence of DNA assembled from overlapping shorter sequences.
106
What is a deoxynucleotide?
An individual monomer (single unit) of DNA.
107
What is a dideoxynucleotide?
An individual monomer of DNA that is missing a hydroxyl group (–OH).
108
What is a DNA microarray?
A method used to detect gene expression by analyzing an array of DNA fragments that are fixed to a glass slide or a silicon chip to identify active genes and identify sequences.
109
What is genome annotation?
The process of attaching biological information to gene sequences.
110
What is a model organism?
A species that is studied and used as a model to understand the biological processes in other species represented by the model organism.
111
What is next-generation sequencing?
A group of automated techniques used for rapid DNA sequencing.
112
What is shotgun sequencing?
A method used to sequence multiple DNA fragments to generate the sequence of a large piece of DNA.
113
What is whole-genome sequencing?
A process that determines the DNA sequence of an entire genome.
114
What is whole-exome sequencing?
Whole-exome sequencing is a lower-cost alternative to whole genome sequencing. In exome sequencing, only the coding, exon-producing regions of the DNA are sequenced.
115
How has whole-exome sequencing been used?
In 2010, whole-exome sequencing was used to save a young boy whose intestines had multiple mysterious abscesses. The child had several colon operations with no relief. Finally, whole-exome sequencing was performed, which revealed a defect in a pathway that controls apoptosis (programmed cell death). A bone-marrow transplant was used to overcome this genetic disorder, leading to a cure for the boy. He was the first person to be successfully treated based on a diagnosis made by whole-exome sequencing.
116
How accessible is human genome sequencing?
Today, human genome sequencing is more readily available and can be completed in a day or two for about $1000.
117
How does the chain termination method work?
The chain termination method involves DNA replication of a single-stranded template with the use of a primer and a regular deoxynucleotide (dNTP), which is a monomer, or a single unit, of DNA. The primer and dNTP are mixed with a small proportion of fluorescently labeled dideoxynucleotides (ddNTPs). The ddNTPs are monomers that are missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to form a chain.
Each ddNTP is labeled with a different color of fluorophore. Every time a ddNTP is incorporated in the growing complementary strand, it terminates the process of DNA replication, which results in multiple short strands of replicated DNA that are each terminated at a different point during replication. When the reaction mixture is processed by gel electrophoresis after being separated into single strands, the multiple newly replicated DNA strands form a ladder because of the differing sizes. Because the ddNTPs are fluorescently labeled, each band on the gel reflects the size of the DNA strand and the ddNTP that terminated the reaction. The different colors of the fluorophore-labeled ddNTPs help identify the ddNTP incorporated at that position. Reading the gel on the basis of the color of each band on the ladder produces the sequence of the template strand.
118
How does shotgun sequencing work?
In shotgun sequencing method, several copies of a DNA fragment are cut randomly into many smaller pieces (somewhat like what happens to a round shot cartridge when fired from a shotgun). All of the segments are then sequenced using the chain-sequencing method. Then, with the help of a computer, the fragments are analyzed to see where their sequences overlap. By matching up overlapping sequences at the end of each fragment, the entire DNA sequence can be reformed. A larger sequence that is assembled from overlapping shorter sequences is called a contig.
119
How does pairwise-end sequencing work?
Originally, shotgun sequencing only analyzed one end of each fragment for overlaps. This was sufficient for sequencing small genomes. However, the desire to sequence larger genomes, such as that of a human, led to the development of double-barrel shotgun sequencing, more formally known as pairwise-end sequencing. In pairwise-end sequencing, both ends of each fragment are analyzed for overlap. Pairwise-end sequencing is, therefore, more cumbersome than shotgun sequencing, but it is easier to reconstruct the sequence because there is more available information.
120
How does next-generation sequencing work?
Since 2005, automated sequencing techniques used by laboratories are under the umbrella of next-generation sequencing, which is a group of automated techniques used for rapid DNA sequencing. These automated low-cost sequencers can generate sequences of hundreds of thousands or millions of short fragments (25 to 500 base pairs) in the span of one day. These sequencers use sophisticated software to get through the cumbersome process of putting all the fragments in order.
121
What is sequence alignment?
A sequence alignment is an arrangement of proteins, DNA, or RNA; it is used to identify regions of similarity between cell types or species, which may indicate conservation of function or structures. Sequence alignments may be used to construct phylogenetic trees. The following website uses a software program called [BLAST (basic local alignment search tool)](http://blast.ncbi.nlm.nih.gov/Blast.cgi).
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What is the history of whole genome sequencing?
The first genome to be completely sequenced was of a bacterial virus, the bacteriophage fx174 (5368 base pairs); this was accomplished by Fred Sanger using shotgun sequencing. Several other organelle and viral genomes were later sequenced. The first organism whose genome was sequenced was the bacterium *Haemophilus influenzae*; this was accomplished by Craig Venter in the 1980s. Approximately 74 different laboratories collaborated on the sequencing of the genome of the yeast *Saccharomyces cerevisiae*, which began in 1989 and was completed in 1996, because it was 60 times bigger than any other genome that had been sequenced. By 1997, the genome sequences of two important model organisms were available: the bacterium *Escherichia coli* K12 and the yeast *Saccharomyces cerevisiae*. Genomes of other model organisms, such as the mouse *Mus musculus*, the fruit fly *Drosophila melanogaster*, the nematode *Caenorhabditis elegans*, and humans *Homo sapiens* are now known.
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What are the benefits of whole genome sequencing?
Having entire genomes sequenced helps with the research efforts in model organisms. The process of attaching biological information to gene sequences is called genome annotation. Annotation of gene sequences helps with basic experiments in molecular biology, such as designing PCR primers and RNA targets.
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How can genomics be used in medicine?
Almost one million genotypic abnormalities can be discovered using DNA microarrays, whereas whole-genome sequencing can provide information about all six billion base pairs in the human genome. Although the study of medical applications of genome sequencing is interesting, this discipline tends to dwell on abnormal gene function. Knowledge of the entire genome will allow future onset diseases and other genetic disorders to be discovered early, which will allow for more informed decisions to be made about lifestyle, medication, and having children. Genomics is still in its infancy, although someday it may become routine to use whole-genome sequencing to screen every newborn to detect genetic abnormalities.
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What are some uses of genomics?
In addition to disease and medicine, genomics can contribute to the development of novel enzymes that convert biomass to biofuel, which results in higher crop and fuel production, and lower cost to the consumer. This knowledge should allow better methods of control over the microbes that are used in the production of biofuels. Genomics could also improve the methods used to monitor the impact of pollutants on ecosystems and help clean up environmental contaminants. Genomics has allowed for the development of agrochemicals and pharmaceuticals that could benefit medical science and agriculture.
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What are some bioethical concerns of genomics?
Knowledge from genomics could be misused, leading to discrimination based on a person's genetics, human genetic engineering, and other ethical concerns. This information could also lead to legal issues regarding health and privacy.
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What is metagenomics?
The study of the collective genomes of multiple species that grow and interact in an environmental niche.
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What is pharmacogenomics?
The study of drug interactions with the genome or proteome; also called toxicogenomics.
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What does it mean to be polygenic?
A phenotypic characteristic caused by two or more genes.
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What is pure culture?
The growth of a single type of cell in the laboratory.
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In what fields is genomics used?
The introduction of DNA sequencing and whole genome sequencing projects, particularly the Human Genome project, has expanded the applicability of DNA sequence information. Genomics is now being used in a wide variety of fields, such as metagenomics, pharmacogenomics, and mitochondrial genomics. The most commonly known application of genomics is to understand and find cures for diseases.
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How can genome analysis be used to predict risk of disease?
Predicting the risk of disease involves screening currently healthy individuals by genome analysis at the individual level. Intervention with lifestyle changes and drugs can be recommended before disease onset. However, this approach is most applicable when the problem resides within a single gene defect. Such defects only account for approximately 5 percent of diseases in developed countries. Most of the common diseases, such as heart disease, are multi-factored or polygenic, and also involve environmental factors such as diet.
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What is an instance of genome analysis used for the risk assessment of disease?
In April 2010, scientists at Stanford University published the genome analysis of a healthy individual (Stephen Quake, a scientist at Stanford University, who had his genome sequenced); the analysis predicted his propensity to acquire various diseases. A risk assessment was performed to analyze Quake’s percentage of risk for 55 different medical conditions. A rare genetic mutation was found, which showed him to be at risk for sudden heart attack. He was also predicted to have a 23 percent risk of developing prostate cancer and a 1.4 percent risk of developing Alzheimer’s. The scientists used databases and several publications to analyze the genomic data.
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What is PCA3?
PCA3 is a gene that is expressed in prostate epithelial cells and overexpressed in cancerous cells. A high concentration of PCA3 in urine is indicative of prostate cancer. The PCA3 test is considered to be a better indicator of cancer than the more well known PSA test, which measures the level of PSA (prostate-specific antigen) in the blood.
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What is an example of a downside of screening for disease?
In 2011, the United States Preventative Services Task Force recommended against using the PSA test to screen healthy men for prostate cancer. Their recommendation is based on evidence that screening does not reduce the risk of death from prostate cancer. Prostate cancer often develops very slowly and does not cause problems, while the cancer treatment can have severe side effects. The PCA3 test is considered to be more accurate, but screening may still result in men who would not have been harmed by the cancer itself suffering side effects from treatment.
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How can pharmacogenomics be used in drug development?
Pharmacogenomics, also called toxicogenomics, involves evaluating the effectiveness and safety of drugs on the basis of information from an individual's genomic sequence. Genomic responses to drugs can be studied using experimental animals (such as laboratory rats or mice) or live cells in the laboratory before embarking on studies with humans. Studying changes in gene expression could provide information about the transcription profile in the presence of the drug, which can be used as an early indicator of the potential for toxic effects. For example, genes involved in cellular growth and controlled cell death, when disturbed, could lead to the growth of cancerous cells. Genome-wide studies can also help to find new genes involved in drug toxicity. Personal genome sequence information can be used to prescribe medications that will be most effective and least toxic on the basis of the individual patient’s genotype. The gene signatures may not be completely accurate, but can be tested further before pathologic symptoms arise.
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How is metagenomics used?
Traditionally, microbiology has been taught with the view that microorganisms are best studied under pure culture conditions, which involves isolating a single type of cell and culturing it in the laboratory. Because microorganisms can go through several generations in a matter of hours, their gene expression profiles adapt to the new laboratory environment very quickly. In addition, the vast majority of bacterial species resist being cultured in isolation. Most microorganisms do not live as isolated entities, but in microbial communities known as biofilms. For all of these reasons, pure culture is not always the best way to study microorganisms. Metagenomics is the study of the collective genomes of multiple species that grow and interact in an environmental niche. Metagenomics can be used to identify new species more rapidly and to analyze the effect of pollutants on the environment.
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How can microbial genomics be used in the creation of biofuels?
Knowledge of the genomics of microorganisms is being used to find better ways to harness biofuels from algae and cyanobacteria. The primary sources of fuel today are coal, oil, wood, and other plant products, such as ethanol. Although plants are renewable resources, there is still a need to find more alternative renewable sources of energy to meet our population’s energy demands. The microbial world is one of the largest resources for genes that encode new enzymes and produce new organic compounds, and it remains largely untapped.
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What are some uses of microbial genomics?
Microorganisms are used to create products, such as enzymes that are used in research, antibiotics, and other anti-microbial mechanisms. Microbial genomics is helping to develop diagnostic tools, improved vaccines, new disease treatments, and advanced environmental cleanup techniques.
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What are some uses of mitochondrial genomics?
Mitochondria are intracellular organelles that contain their own DNA. Mitochondrial DNA mutates at a rapid rate and is often used to study evolutionary relationships. Another feature that makes studying the mitochondrial genome interesting is that the mitochondrial DNA in most multicellular organisms is passed on from the mother during the process of fertilization. For this reason, mitochondrial genomics is often used to trace genealogy.
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How has genomics been used in forensics?
In 2001, the first use of genomics in forensics was published. It was a collaborative attempt between academic research institutions and the FBI to solve the mysterious cases of anthrax communicated via the US Postal Service. Using microbial genomics, researchers determined that a specific strain of anthrax was used in all the mailings.
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How is genomics used in agriculture?
Genomics can reduce the trials and failures involved in scientific research to a certain extent, which could improve the quality and quantity of crop yields in agriculture. Linking traits to genes or gene signatures helps to improve crop breeding to generate hybrids with the most desirable qualities. Scientists use genomic data to identify desirable traits, and then transfer those traits to a different organism. Scientists are discovering how genomics can improve the quality and quantity of agricultural production. For example, scientists could use desirable traits to create a useful product or enhance an existing product, such as making a drought-sensitive crop more tolerant of the dry season.
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What is a biomarker?
An individual protein that is uniquely produced in a diseased state.
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What is a false negative?
An incorrect test result that should have been positive.
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What is the metabolome?
Complete set of metabolites which are related to the genetic makeup of an organism.
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What is metabolomics?
The study of small molecule metabolites found in an organism.
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What is a protein signature?
The set of uniquely expressed proteins in the diseased state.
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What is a proteome?
The entire set of proteins produced by a cell type.
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What is proteomics?
The study of the function of proteomes.
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What is systems biology?
The study of whole biological systems (genomes and proteomes) based on interactions within the system.
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What are the advantages of studying proteomics?
Proteomes can be studied using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins. Proteomics complements genomics and is useful when scientists want to test their hypotheses that were based on genes. Even though all cells of a multicellular organism have the same set of genes, the set of proteins produced in different tissues is different and dependent on gene expression. Thus, the genome is constant, but the proteome varies and is dynamic within an organism. In addition, RNAs can be alternately spliced (cut and pasted to create novel combinations and novel proteins) and many proteins are modified after translation by processes such as proteolytic cleavage, phosphorylation, glycosylation, and ubiquitination. There are also protein-protein interactions, which complicate the study of proteomes. Although the genome provides a blueprint, the final architecture depends on several factors that can change the progression of events that generate the proteome.
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What are the advantages of studying metabolomics?
Metabolomics offers an opportunity to compare genetic makeup and physical characteristics, as well as genetic makeup and environmental factors. The goal of metabolome research is to identify, quantify, and catalogue all of the metabolites that are found in the tissues and fluids of living organisms.
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Which techniques are used for proteomic analysis?
The basic technique for protein analysis is mass spectrometry, but nuclear magnetic resonance (NMR) and protein microarrays are also used.
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How is mass spectrometry used in protein analysis?
The basic technique for protein analysis, analogous to DNA sequencing, is mass spectrometry. Mass spectrometry is used to identify and determine the characteristics of a molecule. Advances in spectrometry have allowed researchers to analyze very small samples of protein. X-ray crystallography, for example, enables scientists to determine the three-dimensional structure of a protein crystal at atomic resolution.
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How is NMR used in protein analysis?
Another protein imaging technique, nuclear magnetic resonance (NMR), uses the magnetic properties of atoms to determine the three-dimensional structure of proteins in aqueous solution.
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How are protein microarrays used in protein analysis?
Protein microarrays have been used to study interactions between proteins. Large-scale adaptations of the basic two-hybrid screen have provided the basis for protein microarrays.
Two-hybrid screening is used to determine whether two proteins interact. In this method, a transcription factor is split into a DNA-binding domain (BD) and an activator domain (AD). The binding domain is able to bind the promoter in the absence of the activator domain, but it does not turn on transcription. A protein called the bait is attached to the BD, and a protein called the prey is attached to the AD. Transcription occurs only if the prey “catches” the bait.
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Why is proteomic analysis difficult?
The challenge of techniques used for proteomic analyses is the difficulty in detecting small quantities of proteins. Although mass spectrometry is good for detecting small amounts of proteins, variations in protein expression in diseased states can be difficult to discern. Proteins are naturally unstable molecules, which makes proteomic analysis much more difficult than genomic analysis.
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How is proteomics used in the detection and treatment of disease?
Genomes and proteomes of patients suffering from specific diseases are being studied to understand the genetic basis of the disease. The most prominent disease being studied with proteomic approaches is cancer. Proteomic approaches are being used to improve screening and early detection of cancer; this is achieved by identifying proteins whose expression is affected by the disease process.
Proteomics is also being used to develop individualized treatment plans, which involves the prediction of whether or not an individual will respond to specific drugs and the side effects that the individual may experience. Proteomics is also being used to predict the possibility of disease recurrence.
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How are biomarkers and protein signatures used in proteomics?
An individual protein is called a biomarker, whereas a set of proteins with altered expression levels is called a protein signature. For a biomarker or protein signature to be useful as a candidate for early screening and detection of a cancer, it must be secreted in body fluids, such as sweat, blood, or urine, such that large-scale screenings can be performed in a non-invasive fashion. The current problem with using biomarkers for the early detection of cancer is the high rate of false-negative results. In other words, many cases of cancer go undetected, which makes biomarkers unreliable. Protein signatures may be more reliable than biomarkers to detect cancer cells.
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What are some examples of biomarkers?
Some examples of protein biomarkers used in cancer detection are CA-125 for ovarian cancer and PSA for prostate cancer.
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What are some programs that use proteomics for cancer detection and treatment?
The National Cancer Institute has developed programs to improve the detection and treatment of cancer. The Clinical Proteomic Technologies for Cancer and the Early Detection Research Network are efforts to identify protein signatures specific to different types of cancers. The Biomedical Proteomics Program is designed to identify protein signatures and design effective therapies for cancer patients.
