Chapter 20: Biotechnology
Describe how a radioactively labeled nucleic acid probe can locate the gene of interest on a multiwell plate. (Use Figure 20.7 in your text to guide your response.)
DNA is immobilized on the membrane is single-stranded and can base pair with any complimentary DNA, excess is rinsed off
The polymerase chain reaction (peR) is a Nobel Prize-winning idea that is used by scientists to amplify DNA, particularly when the quantity of DNA is very small or contaminated. Explain the three initial steps that occur in cycle 1 of PCR.
Denaturation- heat briefly to separate DNA strands 2.Annealing- cool to allow primers to form hydrogen bonds with ends of target sequence 3.extension- DNA polymerase adds nucleotides to the 3' end of each primer
Describe three problems associated with animal cloning.
Often have obesity, pneumonia, liver failure, premature death differentiation doesn't remove all chromatin modifications
what is a restriction fragment
a DNA segment that results from the cutting of a restriction enzyme
What are SNP? How are they used to screen for various diseases?
a single base-pair site where variation is found in at least 1% of the population
electrophoresis
a technique used to separate nucleic acids or proteins that differ in size or electrical charge.
What is the major difference between embryonic stem (ES) cells and adult stem cells?
adult stem cells are not able to give rise to all cell types in an organism
How is a cDNA library formed?
after mRNA is degraded, DNA pol synthesises another strands- 2 wind together
What is the purpose of a Southern blot?
allows us to detect just those bands that include parts of the B-globin gene
What is a totipotent cell?
any cell with the potential to dedifferentiate and then give rise to all the specialized cell types
What are the advantages of using yeasts as hosts for cloning and/or expressing genes of interest?
as easy to grow as bacteria they have plasmids which is rare for eukaryotes
Why does a dideoxyribonucleotide terminate a growing DNA strand?
because it lacks a 3' OH group
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.
can be used to detect the gene's DNA by its ability to base-pair with a complementary sequence on another nucleic acid molecule
what is a genomic library
carries copies of a particular segment from the initial genome
Getting a cloned eukaryotic gene to function in bacterial host cells can be difficult. What are two problems with bacterial gene expression systems, and how is each solved?
certain aspects are different in eukaryotes and bacteria- expression vector is a cloning vector that contains a highly active bacterial promoter just upstream of a restriction site expressing cloned eukaryotic genes in bacteria is the presence of non coding regions- using cDNA
plasmids
circular DNA that replicates separately from the bacterial chromosome
What two techniques discussed earlier in this chapter are used in performing a Southern blot?
combines gel electrophoresis and nucleic acid hybridization
What are two techniques besides use of cloning vectors that can be used to introduce recombinant DNA into eukaryotic cells?
electroporation- brief electrical pulse applied to a solution containing cells created temporary holes in plasma membrane so DNA can enter can inject DNA directly into single eukaryotic cells using microscopically thin needles
what is DNA ligase
glues restriction fragments together
What are genetically modified organisms, and why are they controversial?
has acquired genes by artificial means not safe, environmental consequences
Why are the four nucleotides in DNA each labeled with a different color of fluorescent tag?
indicated the identity of the nucleotide at its end
How does the Ti plasmid make genetic engineering in plants a possibility?
it infects broad leaf plants and inserts itself in the whole plants DNA
what is a bacterial artificial chromosome (BAC)
large plasmids trimmed down so they only contain genes necessary for replication
gene cloning
making multiple copies of a single gene
biotechnology
manipulation of organisms or their components to make useful products
Why is the DNA sample to be separated by gel electrophoresis always loaded at the cathode or negative end of the power source?
nucleic acid molecules carry negative charges on their phosphate groups
What are stem cells?
relatively unspecialized cell that can both reproduce itself indefinitely and under appropriate conditions it can differentiate into specialized cells of one or more types
Explain how transgenic "pharm" animals might be able to produce human proteins.
remove eggs from female and fertilize them in vitro, clone desired gene and inject cloned DNA in nucleus of fertilized egg, some cells can integrate foreign DNA
How is nuclear transplantation performed in animals?
replace the nucleolus of an egg cell with an adult somatic cell, grow the cell, implant the blastocyst into a surrogate mother- first performed on frogs in 1950
Explain the idea of gene therapy, and discuss the problems with this technique as demonstration in the treatment of SCID.
the alteration of an afflicted individual's genes
genetic engineering
the direct manipulation of genes for practical purposes
Describe how short tandem repeats (STRs) can produce a sensitive genetic profile.
the number of repeats present is highly variable from person to person
what is the restriction site
the specific cutting site of restriction enzymes
Explain why shorter DNA molecules travel farther down the gel than larger molecules.
the thicket of agarose fibers impedes larger molecules more than the smaller molecules
what are sticky ends
the uneven ends of restriction fragments
How might induced pluripotent stem (iPS) cells resolve the debate about using stem cells for medical treatments?
they do the same thing ES cells do
What do restriction enzymes do in a normal cell?
they protect the bacterial cell by cutting up foreign DNA. The DNA of the cell is protected by its own RE by the addition of methyl groups to adenines or cytosines within the restriction sites.
How is gene cloning useful? (two ways overall)
to make many copies of a particular gene, or many copies of the protein product it encodes.
Explain the process of gene cloning (4)
1. researchers isolate a plasmid from a bacterial cell and insert foreign DNA, creating a recombinant DNA molecule. 2. The plasmid is returned to a bacterial cell, producing a recombinant bacterium. 3.This single cell reproduces through repeated asexual cell divisions to form a colony of genetically identical cells that all contain the same recombinant plasmid.
How do restriction enzymes work? (3)
1. restriction enzyme cuts the sugar phosphate backbones at individual restriction sites 2. DNA fragment from another source is added: base pairing of sticky ends produces various combinations 3. DNA ligase seals the strands
Give 4 examples of how microarrays are used in understanding patterns of gene expression in cancerous tissue:
1. to classify tumors according to their sites of origin 2.to discover previously unrecognized subtypes of cancer 3. to predict clinical outcome 4. to suggest targets for therapy
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, a microscope slide on which copies of single-stranded DNA fragments from the organism's genes are fixed, with a different gene in each spot 4.Rinse of excess cDNA, scan microarray for fluorescence (yellow) represents gene expressed in tissue sample
What are two potential uses for human iPS cells?
1.disease ridden cells can be model cells for studying 2. patient's own cells can be reprogrammed into iPS cells and used to replace nonfunctional tissue
How many molecules will be produced by four PCR cycles?
16
recombinant DNA
2 strands of DNA engineered to mesh together to make a new strand
what is a cloning vector
A gene carrier/plasmid that transfers DNA from a foreign cell or test tube to another cell
