AP Biology Chp 20-21 Biotechnology

describe how a radioactively labeled nucleic acid probe can locate the gene of interest on a multiwell plate

1. Plate by plate, cells from each well, representing one clone, are transferred to a defined spot on a special nylon membrane. The nylon membrane is treated to break open the cells and denature their DNA: the resulting single-stranded DNA molecules stick to the membrane. 2. The membrane is then incubated in a solution of radioactive probe molecules complementary to the gene of interst. Because the DNA immobilized on the membrane is single-stranded, the single-stranded probe can base-pair with any complimentary DNA on the membrane. Excess DNA is then rinsed off. 3. The membrane is laid under photographic film, allolwing any radioactive areas to expose the film. Black spots on the film correspond to the locations on the membrane of DNA that has hybridized to the probe. Each spot can be traced back to the original well containing the bacterial clone that holds the gene of interest.

getting a cloned eukaryotic gene to function in a bacterial host cells can be difficult. What are two problems with bacterial expression systems and how is each solved

1. certain aspects of gene expression are different in eukaryotes and bacteria. To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active bacterial promoter just upstream of a restriction site where the gene can be inserted in the correct reading frame. 2. Bacterial cells are unable to edit mRNA so genomic DNA presents a problem because of the presence of noncoding regions in most eukaryotic genes. This problem can be surmounted by using a cDNA form of the gene, which only includes the exons.

explain the three initial steps that occur in cycle 1 of PCR

1. denaturation: heat briefly to separate DNA strands 2. annealing: cool to allow primers to form hydrogen bonds with the ends of target sequence 3. extension; DNA polymerase adds nucleotides to the 3' ends of each primer

four steps of DNA microarray assays

1. isolate mRNA 2. make cDNA by reverse transcription, using fluorescently labeled nucleotides 3. apply cDNA mixture to a microarray with different genes in each spot. The cDNA will hybridize with any complementary DNA on the microarray 4.rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample

describe three problems associated with animal clothing

1. many cloned animals exhibit birth defects 2. in the nuclei of fully differentiated cells, a small subset of genes is turned on and expression of the rest is repressed 3. DNA in cells form cloned embryos, like that of differentiated cells, often has more methyl groups than does the DNA in equivalent cells from the normal embryos of the same species

what are two techniques that can be used to introduce recombinant DNA into eukaryotic cells

1.electroporation: a brief electrical pulse applied to a solution containing cells creates temporary holes in their plasma membrane, through which DNA can enter. 2. Scientists can inject DNA direclty into single eukaryotic cells using microscopically thin needles

how many molecules produced by 4 PCR cycles

2^4 = 16

what is the role of DNA ligase

DNA ligase covalently bonds the sugar phosphate backbones of the fragments, whose sticky ends have base-paired

what are SNPs? how are they used to screen for various diseases?

SNPs are single base-pair sites in a genome where nucleotide variation is found in a t least 1% of the population. Scientists use SNPs to screen for various diseases by searching for SNP markers usually inherited with the disease-causing allele.

recombinant DNA

a DNA molecule made in vitro with segments from different sources

what is a cloning vector? Why are bacterial plasmids widely used as cloning vectors?

a DNA molecule that can carry foreign DNA into a host cell and replicate there. Cloning vectors include plasmids and bacterial artificial chromosomes, which move recombinant DNA from a test tube back into a cell, and viruses that transfer recombinant DNA by infection. Bacterial plasmids are widely used as cloining vectors because they can be readily obtained from commercial suppliers, manipulated to form recombinant plasmids, and then introduced into bacterial cells, and they multiply rapidly owing to the high reproductive rate of their host cells.

explain how a bacterial artificial library BAC and a CDNA library are formed

a bacterial artificial library is formed by trimming down the large plasmid to contain just the genes necessary for replicated. cDNA is made in vitro using mRNA as a template for the first strand. Becuase the mRNA contains only exons, the resulting double stranded cDNA carries the complete coding sequence of the gene but not introns

what is the purpose of a genomic library

a genomic library is set of cell clones containing all the DNA segments from a genome, each within a plasmid, BAC, or other cloning vector

define plasmid

a small, circular, double-stranded DNA molecule that carries accessory genes separate from those of a bacterial chromosome; in DNA cloning, used as vectors carrying up to about 10,000 base pairs of DNA. They are also found in some eukaryotes, such as yeasts.

what are stem cells

a stem cell is any relatively unspecialized cell that can produce, during a single division, one identical daughter cell and one more specialized daughter cell that can undergo further differentiation

what is a totipotent cell

a totipotent cell is one that can give rise to all parts of the embryo and adult, as well as extraembryonic membranes in species that have them

what is the major difference between embryonic and adult stem cells

adult stem cells are not able to give rise to all cell types in the organism, though they can generate multiple types. embryonic stem cells are capable of giving rise to differentiated embryonic cells of any type

why is the plasmid engineering with ampR and lacZ

ampR is used because it makes the e coli resistant to the antibiotic ampicillin; lacZ encodes the enzyme which hydrolyzes lactose.

what are genetially modified organisms, and why are they controversial

an organism that has acquired one or more genes by artificial means; also known as a transgenic organism advocates of cautious approach toward GM crops fear that transgenic plants might pass their new genes to close relatives in nearby wild areas

explain why shorter DNA molecules travel farther down the gel than larger molecules

as the nucleic acid molecules move, the thickest of agarose fibers impedes the longer molecules more than it does the shorter ones, separating them by length

why is the DNA sample to be separated by gel electrophoresis always loaded at the cathode or negative end of the power source

because nucleic acid molecules carry negative charges on their phosphate groups, they all travel toward the positive pole in an electric field

purpose of DNA polymerase in DNA sequencing

catalyzes the elongation of new DNA

what are two potential uses for human iPS cells

cells from patients suffering from diseases can be reprogrammed to become iPS cells, which can act as model cells for studying the disease and potential treatment. In the field of regenerative medicine, a patient's own cells could be reprogrammed into iPS cells and then used to replace nonfunctional tissues

why are some colonies blue after the transformation has occured

colonies with nonrecombinant plasmids will be blue because they can hydrolyze x-gal, forming a blue product

why are some of the colonies white after the transformation has occurred

colonies with recombinant plasmids, in which lacZ is disrupted, will be white becuase they cannot hydrolyze x-gal. These plasmids did not take up the hummingbird DNA fragments

explain how microarrays are used in understanding patterns of gene expression in normal and cancerous tissue

different genes are "on" in different tissue types or under different conditions. microarrays can be used to determine what genes are "on" or "off" in a particular type of tumor, and assist in both diagnosis and treatment

how does the Ti plasmid make genetic engineering in plants a possibility

for many plant species, a single tissue cell grown in culture can give rise to an adult plant, meaning that genetic manipulation can be performed on an ordinary somatic cell and the cell then used to generate an organism with new traits. the Ti plasmid integrates a segment of its DNA, known as T DNA, into the chromosomal DNA of its host plant cells

what two techniques discussed earlier in this chapter are used in performing a southern blot

gel electrophoresis and nucleic acid hybridization

explain the idea of gene therapy, and discuss the problems with this technique as demonstrated in the treatment of SCID

gene therapy is the introduction of genes int oan afflicted individual for therapeutic purposes. The problems witj this treatment were demonstrated during the trial treatment of SCID, when three patients developed lukemia. Two factors may have contributed to the development of lukemia: the insertion of the retroviral vector near a gene involved in the proliferation of blood cells and an unknown function of the replacement gene itself

describe how short tandem repeats STRs can produce a sensitive genetic profile

in short tandem repeats, simple sequence DNA containing multiple tandemly repeated units of two to five nucleotides. variations in STR act as genetic markers in STR analysis, used to prepare genetic profiles. the number of repeat prsent in these regions in highly variable from person to person, creating a more sensitive genetic profile

why does a dideoxyribonucleotide terminate a growing DNA strand?

new DNA elongates in a 5 to 3 direction, and dideoxyribonucleotides lacks the 3 - OH group to continue the chain

once the hummingbird DNA is cloned, we have the problem of finding the piece of DNA that holds our gene of interest. Explain how nucleic acid hybridization will accomplish this task

nucleic acid hybridization is used to detect the gene's DNA by its ability to base-pair with a complementary sequence on another nucleic acid molecule

purpose of the template strand in DNA sequencing

provides pattern for the DNA strand to be synthesized

how might induced pluripotent stem cells resolve the debate about using stem cells for medical treatments

researchers have been able to reprogram differentiated cells to act like ES cells, resolving the ethical and political issues surrounding harvesting ES cells and human embryos

explain how transgenic pharm animals might be able to produce human proteins

scientists can introduce a gene from an animal or one genotype into the genome of another individual, often of a different species. Assuming that the introduced gene encodes a protein desired in large quantities, these transgenic animals can act as pharmaceutical "factories"

how is nuclear transplantation performed in animals

scientists remove the nucleus of an unfertilized or fertilized egg, and replace it with the nucleus of a differentiated cell

what is the purpose of a southern blot

southern blotting is a technique that enables specific nucleotide sequences to be detected in samples of DNA. It involves gel electrophoresis of DNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe

gel electrophoresis

technique used to separate nucleic acids or proteins that differ in size or electrical charge

why are both the gene of interest and the plasmid cut with the same restriction enzyme

the are cut with the same restriction enzyme so that both the gene and the plasmid will have the same sticky ends and be able to be annealed by ligase

a patient who is a carrier for sickle-cell anemia would have a gel electrophoresis pattern showing four bands. add this pattern to your gel and explain why the gel shows a four band pattern

the carrier is heterozygous and therefore has both alleles. the normal allele will produce three fragments. the abnormal allele will produce two fragments, one of which is the same as the normal allele. therefore, there will be four different size fragments.

why are the four nucleotides in DNA each labeled with a different color of fluorescent tag

the color of the florescent tag on each strand indicates the identity of the nucleotide at its end

genetic engineering

the direct manipulation of genes for practical purposes

biotechnology

the manipulation of organisms or their components to produce useful products. It includes selective breeding, genetic engineering, and methods of DNA analysis

gene cloning

the production of multiple copies of a single gene

purpose of deoxyribonucleotides in DNA sequences

these are raw materials for the synthesizing of the new strand

purpose of dideoxyribonucleotides in DNA sequences

these nucleotide forms are added randomly, and when this occurs, prevent further elongation of the strand

purpose of the primer in DNA sequencing

will bind to the DNA and serve as the point where DNA polymerase can begin synthesis

why are the advantages of using yeasts as host for cloning and/or expressing genes of interest

yeasts are easy to grow as bacteria and they have plasmids