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Ch 12 practice test, Biology Ch. 12 - DNA Technology and Genomics, Chapter 12, Chapter 12, Human Genome Project, BIO TEST CH 12, last bio

Terms in this set (351)

DNA technology is a set of methods for studying and manipulating genetic material. DNA
technology has many useful applications in our world today, including providing evidence for
court proceedings, engineering food crops, and producing organisms tailor-made for specific
purposes. In fact, DNA technology is one of the hottest areas of research in all of biology.
Many techniques of DNA technology rely on recombinant DNA. Recombinant DNA is DNA
made by combining genes from different sources, often different species. Recombinant DNA
technologies rely on bacteria. Many bacteria contain plasmids, small circular pieces of DNA that
are separate from the bacteria's larger chromosome. Plasmids can be cut open with restriction
enzymes, proteins that cut DNA at very specific nucleotide sequences. Once a plasmid is
snipped open by a restriction enzyme, one or more genes of interest can be inserted into it. After
pasting the DNA back together, this plasmid contains one or more foreign genes. In other words,
the plasmid is now a piece of recombinant DNA. If the recombinant plasmid is then inserted into
a host bacterium, that bacterium is now considered a genetically modified organism. If the
foreign DNA is from a different species, the bacterium is called a transgenic organism.
Bacterial plasmids are often used in recombinant DNA experiments because bacteria reproduce
very quickly and thus can rapidly make more copies of the gene of interest.
Let's think of an analogy to further illustrate the technique of recombinant DNA technology.
Imagine that you have a ring that is too small for your finger. A jeweler can enlarge the ring by
cutting it and inserting a small piece of metal into the opening. The ring is then heated so that the
ends melt together, forming a complete ring again that is now larger in size. Typically, a jeweler
will match the color and material of a ring as closely as possible so that the extension is not
noticed. However, any type of metal could be inserted, including pieces of a different material or
color, making a transgenic ring!
A second DNA technology is DNA fingerprinting, also called DNA profiling. DNA profiling is
the analysis of DNA to determine whether it comes from a certain individual. Such methods can
be applied to medical research and to forensics, the scientific study of evidence for legal
proceedings. To understand DNA profiling, you need to understand several techniques and
concepts. To help you keep track, let's imagine that you are attempting to solve a murder case.
Are you ready for some detective work?
In this case, you recovered a few skin cells from the undersides of a victim's fingernails.
Presumably, these cells came from the victim's attacker. You would like to compare the DNA in
these skin cells with the DNA of a suspect who is in custody. However, there are not many skin
cells to work with. So the first thing you need to do is to make more copies of the DNA. You can
do this with a technique called the polymerase chain reaction. The polymerase chain reaction ,
abbreviated PCR, is a method for quickly and precisely copying a small amount of DNA into a
large sample. PCR proceeds by heating double-stranded DNA in order to separate the two
strands. Each separated strand is then copied. This cycle is repeated, with the number of DNA
molecules doubling after every cycle. Within just a few hours, PCR can produce billions of
strands of DNA from a small initial sample. Now you have enough DNA to continue your
investigation.
The next step is to compare the DNA from the victim's fingernails with the DNA of the suspect.
To do this, forensic scientists compare specific segments of DNA, referred to as short tandem
repeats. Short tandem repeats are very short sequences, usually just a few base pairs long, which
are repeated many times in a row. The specific number of repetitions varies between individuals.
For example, I might have a spot on my DNA that contains the sequence AGAT repeated 13
times, while you might have that sequence repeated 22 times at that spot. Because short tandem
repeats vary widely from person to person, they make excellent targets for comparison.
But how do you actually compare the length of short tandem repeats between two samples? This
brings us to our last DNA technology technique, gel electrophoresis. Gel electrophoresis is a
method that can separate DNA fragments based on length. If you recall the structure of DNA,
you may remember that DNA is negatively charged due to the negatively charged phosphate
groups in the sugar-phosphate backbone. In gel electrophoresis, a sample of DNA is placed at
one end of a slab of gel, which is like thick Jell-O. A battery is hooked up to the gel, with the
negative end near the DNA and the positive end at the other end of the gel. Because opposite
charges attract, the negatively charged DNA will move through the gel toward the positive
electrode. However, not all DNA will move at the same speed. The longer the DNA fragment,
the more time it will take to pass through the thick gel. After a given period of time, short DNA
fragments will have migrated further through the gel than long DNA fragments.
Thus, using gel electrophoresis, you can compare the lengths of short tandem repeats between
your two DNA samples. After you obtain the short tandem repeats from both samples, you run
them out on a gel. You can then stain the gel to make the DNA fragments visible. If the
fragments from the two samples match, this proves that the DNA from under the victim's
fingernails matches the DNA from the suspect. This is strong evidence of the guilt of your
suspect. Of course, DNA profiling can provide equally strong evidence of innocence. In this
case, the DNA profiles match, and you have solved the case. Good work!
Let's review with a brief quiz. Here's how it works. I'll make a statement. If the sentence is true,
repeat it out loud. If the statement is false, correct it and say the correct statement out loud.
Follow me? Trust me, saying the words out loud will help you remember them better. I do it all
the time.
Let's give it a try:
Recombinant DNA is made by combining two pieces of DNA from the same source...
That's false. Recombinant DNA is made by combining two pieces of DNA from different
sources.
Restriction enzymes cut DNA at specific nucleotide sequences...
That's true. Restriction enzymes cut DNA at specific nucleotide sequences.
The polymerase chain reaction is a method for elongating a piece of DNA...
That's false. The polymerase chain reaction is a method for copying a piece of DNA.
Gel electrophoresis separates DNA molecules based on length...
That's true. Gel electrophoresis separates DNA molecules based on length.
The technique of nucleic acid hybridization is commonly used to identify specific DNAs in complex mixtures such as genomic libraries. Hybridization process takes advantage of the specificity of base-pairing between the two strands of nucleic acids. Molecular biologists can take advantage of this feature to use a known, specific sequence to find its partner in a complex mixture. Any single-stranded sequence of nucleic acid - DNA or RNA - can be tagged with a radioactive label or with another detectable label, such as a fluorescent dye. Identifying the sequence of the RNA or DNA probe depends on knowledge of at least part of the sequence of the gene of interest.


This labeled sequence can then be used as a probe to identify its complement in a complex mixture. The renaturing is termed hybridization because of the combination of labeled probe and unlabeled nucleic acid for a hybrid segment through base pairing.

A method called Southern blotting combines gel electrophoresis with nucleic acid hybridization. Because gel electrophoresis yields too many bands to distinguish individually, scientists use DNA hybridization with a specific probe to label discrete bands that derive from the gene of interest. Labeled nucleic acid probes that hybridize with mRNAs can provide information about the time or place in the organism at which a gene is transcribed. In the method called Northern Blotting, scientists carry out gel electrophoresis on samples of RNA then proceed with an RNA hybridization to locate a sequence of interest. Northern blotting is a play on words based on the method's similarity to Southern blotting.
Plants are easier to genetically engineer than most animals because a single tissue cell grown in culture and genetically manipulated can give rise to an adult plant with new traits. The most commonly used vector for introducing new genes into plant cells is a plasmid, called the Ti plasmid, from the soil bacterium Agrobacterium tumefaciens. Genes conferring useful traits, such as pest resistance, herbicide resistance, delayed ripening, and increased nutritional value, can be transferred from one plant variety or species to another using the Ti plasmid.

The Ti plasmid is isolated from the bacterium Agrobacterium tumefaceins. The segment of the plasmid that integrates into the genome of the host cells is called T DNA. The foreign gene of interest is inserted into the middle of the T DNA using restriction enzymes and DNA ligase. This results in a recombinant Ti plasmid.

Scientists can introduce recombinant Ti plasmids into plant cells by electroporation or by infecting plant cells in culture with bacteria containing the recombinant plasmid. Alternatively, plasmids can be returned to Agrobacterium, which is then applied as a liquid suspension to the leaves of susceptible plants, infecting them. Once a plasmid is taken into a plant cell, its T DNA integrates into the cell's chromosomal DNA.

Transformed cells carrying the transgene of interest can regenerate complete plants that exhibit the new trait conferred by the transgene. Genetic engineering is rapidly replacing traditional plant-breeding programs for simple genetic traits such as herbicide or pest resistance. Genetically engineered crops that can resist destructive insects have reduced the need for chemical insecticides. In India, the insertion of a salinity-resistance gene from a coastal mangrove plant into the genomes of several rice varieties has resulted in rice plants that can grow in water three times as salty as seawater. The research foundation that carried out this genetic engineering estimates that one-third of all irrigated land has high salinity owing to over-irrigation and intensive use of chemical fertilizers, which represents a serious threat to the food supply. Salinity-resistant crop plants would be enormously valuable worldwide.

Genetic engineering also has great potential for improving the nutritional value of crop plants. For instance, scientists have developed transgenic rice plants that produce yellow rice grains containing beta-carotene, which our body uses to make vitamin A. This "golden" rice could help prevent vitamin A deficiency in the half of the world's population that depends on rice as a staple food.
Gene therapy—introducing genes into an afflicted individual for therapeutic purposes—holds great potential for treating genetic disorders caused by a single gene. In theory, a normal allele of the defective gene could be inserted into dividing somatic cells of the tissue affected by the disorder, such as bone marrow cells. A retrovirus that has been rendered harmless is used as a vector in this procedure, which exploits the ability of a retrovirus to insert a DNA transcript of its RNA genome into the chromosomal DNA of its host cell.



Once the normal gene has been inserted into the retrovirus, the virus is allowed to infect the bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into the chromosome.


The engineered cells are injected into the patient. If the foreign gene carried by the retroviral vector is expressed, the cell and its descendants will possess the gene product, and the patient may be cured.

Gene therapy also raises technical questions. How can the activity of the transferred gene be controlled so that cells make appropriate amounts of the gene product at the right time and in the right place? How can scientists be sure that the insertion of the therapeutic gene does not harm some other necessary cell function?

In addition to these technical challenges, gene therapy raises ethical questions. Some critics believe that tampering with human genes in any way is immoral. Others see no difference between the transplantation of genes into somatic cells and the transplantation of organs. The treatment of human germ-line cells in the hope of correcting a defect in future generations raises ethical questions. Under what circumstances, if any, should we alter the genomes of human germ lines? Would this inevitably lead to the practice of eugenics, a deliberate effort to control the genetic makeup of human populations? From a biological perspective, the elimination of unwanted alleles from the gene pool could be problematic because genetic variation is a necessary ingredient for the survival of a species as environmental conditions change with time. Genes that are damaging under some conditions may be advantageous under other conditions. Do we have the right to make genetic changes that could be detrimental to the survival of our species in the future?
Body fluids or small pieces of tissue may be left at the scene of a violent crime or on the clothes of the victim or assailant. If enough blood, semen, or tissue is available, forensic laboratories can determine the blood type or tissue type by using antibodies to detect specific cell-surface proteins. Such tests require fairly fresh samples in relatively large amounts, however, and can serve only to exclude a particular suspect. DNA testing, in contrast, can identify the guilty individual with a high degree of certainty because the DNA sequence of every person is unique (except for identical twins). Genetic markers that vary in the population can be analyzed for a given person to determine that individual's unique set of genetic markers, or genetic profile. This term is preferred over DNA fingerprinting by forensic scientists, who want to emphasize the heritable aspect of these markers rather than simply the fact that they produce a pattern on a gel that, like a fingerprint, is visually recognizable.

The FBI started applying DNA technology in forensics in 1988, using RFLP analysis by Southern blotting to detect similarities and differences in DNA samples. This method required much smaller samples of blood or tissue than earlier methods—only about 1,000 cells. Today, in place of RFLPs, forensic scientists usually use an even more sensitive method, which takes advantage of genetic markers called short tandem repeats (STRs). STRs are variations in the lengths of certain repeated base sequences in specific regions of the genome. The number of repeats present in these regions is highly polymorphic, varying from person to person. For example, one individual may have the sequence AGAT repeated 30 times at one genome locus, whereas another individual may have 18 repeats at this locus.

PCR is used to amplify particular STRs using sets of primers that are labeled with different colored fluorescent tags; the length of the region, and thus the number of repeats, can then be determined by electrophoresis. Since Southern blotting is not required, this method is quicker than RFLP analysis. The PCR step allows the method to be used even when the DNA is in poor condition or available in only minute quantities. A tissue sample containing as few as 20 cells can be sufficient for PCR amplification. In a murder case, for example, this method can be used to compare DNA samples from the suspect, the victim, and a small amount of blood found at the crime scene.

Forensic scientists test only a few selected portions of the DNA—about 13 STR markers. The probability that two people (who are not identical twins) would have exactly the same set of STR markers is vanishingly small. The Innocence Project, a nonprofit organization dedicated to overturning wrongful convictions, uses STR analysis of archived samples from crime scenes to revisit old cases. As of 2006, 18 innocent people had been released from prison as a result of forensic and legal work by this group. In another example of genetic profiling, a comparison of the DNA of a mother, her child, and the purported father can conclusively settle a question of paternity. Sometimes paternity is of historical interest: Genetic profiles provided strong evidence that Thomas Jefferson or one of his close male relatives fathered at least one of the children of his slave, Sally Hemings. Analyzing genetic profiles can also identify victims of mass casualties. After the World Trade Center attack in 2001, more than 10,000 samples of victims' remains were compared with DNA traces on personal items provided by families. These comparisons led to the identification of nearly 3,000 victims.

Just how reliable is a genetic profile? The greater the number of markers examined in a DNA sample, the more likely it is that the profile is unique to one individual. In forensic cases using STR analysis with 13 markers, the probability of two people having identical DNA profiles is somewhere between one chance in 10 billion and one in several trillion. The exact probability depends on the frequency of those markers in the general population. Information on how common various markers are in different ethnic groups is critical because these marker frequencies may vary considerably among ethnic groups and between a particular ethnic group and the population as a whole. With the increasing availability of frequency data, forensic scientists can make extremely accurate statistical calculations. Genetic profiles are now accepted as compelling evidence by legal experts and scientists alike.
Gel electrophoresis separates macromolecules—
nucleic acids or proteins—on the basis of their rate of movement through a polymer gel in an electrical field. The rate of movement of each molecule depends on its size, electrical charge, and other physical properties. In restriction fragment analysis, the DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis.

When the mixture of restriction fragments from a particular DNA molecule undergoes electrophoresis, it yields a band pattern characteristic of the starting molecule and the restriction enzyme used. The relatively small DNA molecules of viruses and plasmids can be identified simply by their restriction fragment patterns. The separated fragments can be recovered undamaged from gels, providing pure samples of individual fragments.

Scientists can use restriction fragment analysis to compare two different DNA molecules representing, for example, different alleles of a gene. Because the two alleles differ slightly in DNA sequence, they may differ in one or more restriction sites. If the alleles do differ in restriction sites, each produces different-sized fragments when digested by the same restriction enzyme. In gel electrophoresis, the restriction fragments from the two alleles produce different band patterns, allowing researchers to distinguish the two alleles. Restriction fragment analysis is sensitive enough to distinguish between two alleles of a gene that differ by only one base pair in a restriction site, such as the normal and sickle-cell alleles of the β-globin gene.
Often, it is important to determine the length of each DNA fragment on an electrophoresis. This may be easily done if a marker DNA sample is run alongside the experimental samples on the gel. The marker DNA sample contains a number of DNA molecules, each with a known molecular size. Molecular size is measured in terms of number of base pairs in a DNA fragment. Our marker DNA includes 8 DNA fragments that have 23130, 9416, 6557, 4361, 2322, 2027, 564, and 125 base pairs. (The 125 base pair fragment is often not seen in experiments, either because it is too faint or because it runs off the gel.) This illustration shows an example of a gel with this DNA marker and some "unknown" DNA samples (DNA I and DNA II).

To determine sizes of unknown DNA fragments, a calibration curve is first made of the known marker DNA data. For each fragment, the y-axis shows the log of the number of base pairs (bp) and the x-axis shows the distance that fragment traveled in millimeters (mm). A new calibration curve must be made for each gel because the exact migration distances of marker DNA fragments vary from experiment to experiment.

Next, the migration distances for the unknown DNA bands are measured and the sizes of the DNA fragments are read from the calibration curve, as illustrated here.

To determine the length of a fragment in DNA II, click on the orange band that represents the fragment. A line will be drawn from the distance the fragment traveled (on the x-axis) to the calibration curve. At the point that it intersects the calibration curve, another line will be drawn to the value for the length in base pairs of the fragment. To determine the length of the DNA fragments in DNA I, drag the ruler down until it is directly above DNA I. Click on each orange band in DNA I to measure the fragment's length in base pairs.
The presence of an abnormal allele can be diagnosed with reasonable accuracy if a closely linked genetic marker has been found. A genetic marker is a DNA sequence that varies in a population; in a gene, such sequence variation is the basis of different alleles. Just like coding sequences, noncoding DNA at a specific place or locus on a chromosome may exhibit small nucleotide differences among individuals, or polymorphisms. Single base-pair variations in the genomes of the human population serve as useful genetic markers. A single base-pair site where variation is found in at least 1% of the population is called a single nucleotide polymorphism (SNP). SNPs occur on average about once in 100 to 300 base pairs in the human genome, in both coding and noncoding sequences.

Some SNPs alter the sequence recognized by a restriction enzyme, changing the lengths of the restriction fragments formed by digestion with that enzyme. This type of sequence change is called a restriction fragment length polymorphism (RFLP, pronounced "Rif-lip").

Today, SNPs can be detected by very sensitive microarray analysis or by PCR. The presence of an abnormal allele that causes a genetic disorder can be diagnosed with reasonable accuracy if a closely linked SNP marker has been found. Alleles for Huntington's disease and a number of other genetic diseases were first detected by means of RFLPs in this indirect way. If the marker and the gene itself are close enough, crossing over between the marker and the gene is very unlikely to occur during gamete formation, and the two regions are almost always inherited together.
Chapter 12 DNA Technology and Genomics
biotechnology- The use of living organisms (often microbes) to perform
useful tasks; today, usually involves DNA technology.
clone- As a verb, to produce genetically identical copies of a cell,
organism, or DNA molecule. As a noun, the collection of cells, organisms,
or molecules resulting form cloning; also (colloquially), a single organism
that is genetically identical to another because it arose from the cloning of a
somatic cell.
complementary DNA (cDNA)- A DNA molecule made in vitro using mRNA
as a template and the enzyme reverse transcriptase. A cDNA molecule
therefore corresponds to a gene but lacks the introns present in the DNA of
the genome.
DNA fingerprinting- See DNA profiling.
DNA ligase- An enzyme, essential for DNA replication, that catalyzes the
covalent bonding of adjacent DNA strands; used in genetic engineering to
paste a specific piece of DNA containing a gene of interest into a bacterial
plasmid or other vector.
DNA technology- Methods used to study and/or manipulate DNA,
including recombinant DNA technology.
forensics- The scientific analysis of evidence for crime scene and other
legal proceedings. Also referred to as forensic science.
FOXP2 gene- gene in humans that may play a role in the ability of humans
to communicate by speech.
gel electrophoresis- A technique for separating and purifying
macromolecules, either DNA's or proteins. A mixture of the
macromolecules is placed on a gel between a positively charged electrode
and a negatively charged one. Negative charges on the molecules are
attracted to the positive electrode, and the molecules migrate toward that
electrode. The molecules separate in the gel according to their rates of
migration, which is mostly determined by their size.
Chapter 12 page 2
gene cloning- The production of multiple copies of a gene.
gene therapy- A treatment for a disease in which the patient's defective
gene is supplemented or altered.
genetic engineering- The direct manipulation of genes for practical
purposes.
genetically modified (GM) organism- An organism that has acquired one
or more genes by artificial means. If the gene is from another species, the
organism is also known as a transgenic.
genomic library- A set of DNA segments representing an organism's
entire genome; each segment is usually carried by a plasmid or phage.
genomics- The study of whole sets of genes and their interactions.
Human Genome Project (HGP)- An international collaborative effort to
map and sequence the DNA of the entire human genome.
nucleic acid probe- In DNA technology, a labeled single-stranded nucleic
acid molecule used to find a specific gene or other nucleotide sequence
within a mass of DNA. The probe hydrogen-bonds to the complementary
sequence in the targeted DNA.
plasmid- A small ring of independently replicating DNA separate from the
main chromosome(s). Plasmids are found in prokaryotes and yeast.
polymerase chain reaction(PCR)- A technique used to obtain may copies
of a DNA molecule or part of a DNA molecule. A small amount of DNA
mixed with a heat-resistant DNA polymerase, DNA nucleotides, and a few
other ingredients replicates repeatedly in a test tube.
primers- Short, artificially created, single-stranded DNA molecules that
bind to each end of a target sequence to drive a PCR procedure.
proteomics- The study of whole sets of proteins and their interactions.
Chapter 12 page 3
recombinant DNA- A DNA molecule carrying genes derived from two or
more sources.
repetitive DNA- Nucleotide sequences that are present in many copies in
the DNA of a genome. The repeated sequences may be long or short and
may be located next to each other (tandomly) or dispersed in the DNA.
restriction enzyme- A bacterial enzyme that cuts up foreign DNA (at
specific restriction sites), thus protecting bacteria against intruding DNA
from phages and other organisms. Restriction enzymes are used in DNA
technology to cut DNA molecules in reproducible ways.
restriction fragments- Molecules of DNA produced from a longer DNA
molecule cut up by a restriction enzyme; used in genome mapping and
other applications.
restriction fragment length polymorphism (RFLP)- The differences in
homologous DNA sequences that are reflected in different lengths of
restriction fragments produced when the DNA is cut up with restriction
enzymes.
restriction site- A specific sequence on a DNA strand that is recognized
as a "cut site" by a restriction enzyme.
reverse transcriptase- An enzyme used by retroviruses that catalyzes the
synthesis of DNA on an RNA template.
SNP (single nucleotide polymorphism)- A variation in DNA sequence
found within the genomes of at least 1% of a population.
STR analysis (short tandem repeat analysis)- A method of DNA profiling
that involves the comparison of the lengths of short tandem repeat (STR)
sequences selected from specific sites within the genome.
STRs (short tandem repeats)
telomere- The repetitive DNA at each end of a eukaryotic chromosome.
Chapter 12 page 4
Ti plasmid- A bacterial plasmid that induces tumors in plant cells that the
bacterium infects; often used as a vector to introduce new genes into plant
cells. Ti stands for tumor-inducing.
transgenic organism- An organism that contains genes from another
species.
transposable element- A transposable genetic element, or "jumping
gene"; a segment of DNA that can move from one site to another within a
cell and serve as an agent of genetic change.
vaccine- A harmless variant or derivative of a pathogen used to stimulate a
host organism's immune system to mount a long-term defense against the
pathogen.
vector- In molecular biology, a piece of DNA, usually a plasmid or a viral
genome, that is used to move genes from one cell to another.
whole-genome shotgun method- A method for determining the DNA
sequence of an entire genome. After a genome is cut into small fragments,
each fragment is sequenced and then placed in the proper order.