Study Guide Lec 15 Flashcards

1
Q

True or false?
Molecular genetics has had profound effects on all fields of biology. Whole genomes have been sequenced, structures of genes elucidated, the patterns of molecular evolution studied.

A

true

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2
Q

How is Recombinant DNA technology being used?

A

to diagnose and screen for genetic diseases, and gene therapy is being explored.

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3
Q

___ DNA is used to make pharmaceutical products, such as recombinant insulin and clotting factors. Genetically modified organisms is changing the lives of farmers and improving agricultural productivity and the quality of food and fiber.

A

Recombinant

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4
Q

What feature is commonly seen in the sequences recognized by type II restriction enzymes?

A

The recognition sequences are palindromic, and 4−8 base pairs long.

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5
Q

What normal role do restriction enzymes play in bacteria?

A

Restriction enzymes cut foreign DNA, such as viral DNA, into fragments.

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6
Q

How do bacteria protect their own DNA from the action of restriction enzymes?

A

Bacteria protect their own DNA by modifying bases, usually by methylation, at the recognition sites.

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7
Q

Explain how gel electrophoresis is used to separate DNA fragments of different lengths.

A

Gel electrophoresis uses an electric field to drive DNA molecules through a gel that acts as a molecular sieve. The gel is an aqueous matrix of agarose or polyacrylamide. DNA molecules are loaded into a slot or well at one end of the gel. When an electric field is applied, the negatively charged DNA molecules migrate toward the positive electrode. Shorter DNA molecules are less hindered by the agarose or polyacrylamide matrix and migrate faster than longer DNA molecules, which must wind their way around obstacles and through the pores in the gel matrix.

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8
Q

After DNA fragments have been separated by gel electrophoresis, how can they be visualized?

A

DNA molecules can be visualized by staining with a fluorescent dye, such as ethidium bromide, that intercalates between the stacked bases of the DNA double helix, and the dye–DNA complex fluoresces when irradiated with an ultraviolet light source. Alternatively, they can be visualized by attaching radioactive or chemical labels to the DNA before it is placed in the gel.

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9
Q

___ ___ is used to detect and visualize specific DNA fragments that have a sequence complementary to a labeled DNA probe.

A

southern blotting

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10
Q

Describe the process of southern blotting

A

DNA is first cleaved into fragments with restriction endonucleases. The fragments are separated by size via gel electrophoresis. These fragments are then denatured and transferred by blotting onto the surface of a membrane filter. The membrane filter now has single-stranded DNA fragments bound to its surface, separated by size as in the gel. The filter is then incubated with a solution containing a denatured, labeled probe DNA. The probe DNA hybridizes to its complementary DNA on the filter. After washing away excess unbound probes, the labeled probe hybridized to the DNA on the filter can be detected using the appropriate methods to visualize the label. For radioactively labeled probes, the bound probe is detected by exposure to X- ray film. Other probe labeling methods detect bound probe using enzymatic reactions that generate luminescence or color.

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11
Q

Give three important characteristics of cloning vectors.

A

(1) An origin of DNA replication so they can be maintained in a cell
(2) A gene, such as antibiotic resistance, to select for cells that carry the vector
(3) A unique restriction site or series of sites to where a foreign DNA molecule may be inserted

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12
Q

Name the following type of cloning:

Vector and foreign DNA are cut with the same restriction enzyme, then ligated together with DNA ligase.

A

Restriction cloning

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13
Q

Name the following type of cloning:
DNA fragments generated by PCR may be ligated to plasmid vectors in either of two ways. One way is to synthesize PCR primers that have restriction sites at or near their 5′ ends. The resulting PCR fragments can be digested with the appropriate restriction enzymes to generate sticky ends for restriction cloning

A

PCR fragment cloning

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14
Q

Briefly explain how an antibiotic-resistance gene and the lacZ gene can be used to determine which cells contain a particular plasmid.

A

Many plasmids designed as cloning vectors carry a gene for antibiotic resistance and the lacZ gene. The lacZ gene on the plasmid has been engineered to contain multiple unique restriction sites. Foreign DNAs are inserted into one of the unique restriction sites in the lacZ gene of plasmids and the plasmids are transformed into E. coli cells lacking a functional lacZ gene. Transformed cells are plated on a medium containing the appropriate antibiotic to select for cells that carry the plasmid. The medium also contains an inducer for the lac operon, so the cells express the lacZ gene, and X-gal, a substrate for beta-galactosidase that will turn blue when cleaved by β-galactosidase. The colonies that carry plasmid without foreign DNA inserts will have intact lacZ genes, make functional β-galactosidase, cleave X-gal, and turn blue. Colonies that carry plasmid with foreign DNA inserts will not make functional β-galactosidase because the lacZ gene is disrupted by the foreign DNA insert. They will remain white. Thus, cells carrying plasmids with inserts will form white colonies. This is known as blue/white selection.

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15
Q

Describe the process of PCR

A

First, the double-stranded template DNA is denatured by high temperature. Then, primers corresponding to the ends of the DNA sequence to be amplified are annealed to the single-stranded DNA template strands. These primers are extended by a thermostable DNA polymerase so that the target DNA sequence is duplicated. These steps are repeated 30 times or more. Each cycle of denaturation, primer annealing, and extension results in doubling the number of copies of the target sequence between the primers.

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16
Q

Name four limitations of PCR

A

1) sequence of the gene to be amplified must be known in order to synthesize primers
2) extreme sensitivity of the technique renders it susceptible to contamination
3) the most common thermostable DNA polymerase (Taq) has a relatively high error rate
4) amplification is usually limited to fragments of up to a few thousand bp

17
Q

What is real-time PCR?

A

Real-time PCR uses fluorescent probes to detect the formation of specific PCR products, and a sensitive instrument to quantify the amount of PCR product formed after each cycle of PCR, while the reaction is proceeding. This technique is used to quantify absolute or relative amounts of template DNA in samples.

18
Q

A ___ library is generated by cloning fragments of chromosomal DNA into a cloning vector. Chromosomal DNA is randomly fragmented by shearing or by partial digestion with a restriction enzyme.

A

genomic

19
Q

A ____ library is made from mRNA sequences. Cellular mRNAs are isolated and then reverse transcriptase is used to copy the mRNA sequences to cDNA, which are cloned into plasmid or phage vectors.

A

cDNA

20
Q

A cDNA library only represents the genes expressed where?

A

in the tissues of origin for the RNA.

21
Q

____ hybridization involves hybridization of radiolabeled or fluorescently labeled DNA or RNA probes to DNA or RNA molecules that are still in the cell.

A

situ

22
Q

What is situ hybridization used for?

A

o visualize the expression of specific mRNAs in different cells and tissues and the location of genes on metaphase or polytene chromosomes.

23
Q

What is the purpose of the dideoxynucleoside triphosphate in the dideoxy sequencing reaction?

A

Dideoxynucleoside triphosphates (ddNTPs) act as a substrate for DNA polymerase but cause termination of DNA synthesis when they are incorporated, because they lack the 3′–OH for the addition of the next nucleotide. Mixed with regular dNTPs, fluorescently labeled ddNTPs generate a series of DNA fragments that have terminated at every nucleotide position along the template DNA molecule being sequenced. These fragments can be separated by gel electrophoresis. Because each of the four ddNTPs carries a different fluorescent label, a laser detector scanning near the end of the gel can distinguish which base terminates each fragment. Reading the fragments from shorter to longer, an automated DNA sequencer can determine the sequence of the template DNA molecule.

24
Q

What is DNA fingerprinting and what is it used for?

A

DNA fingerprinting is the typing of an individual for genetic markers at highly variable loci. This is useful for forensic investigations, to determine whether the suspect could have contributed to the evidentiary DNA obtained from blood or other bodily fluids found at the scene of a crime. Other applications include paternity testing and the identification of bodily remains.

25
Q

What types of sequences are examined in DNA fingerprinting?

A

The first loci used for DNA fingerprinting were variable number of tandem repeat (VNTR) loci; these consist of short tandem repeat sequences located in introns or spacer regions between genes. The number of repeat sequences at the locus does not affect the phenotype of the individual in any discernible way, so the numbers of repeats at each of these loci are highly variable in the population. More recently, loci with smaller repeat sequences of just a few nucleotides, called short tandem repeats (STRs), have been adopted because they can be amplified by PCR. The variable number of repeats creates PCR fragments of different sizes. Genotyping at 13 to 15 of these unlinked STR loci can identify one individual among trillions of potential genotypes.

26
Q

Briefly explain how site-directed mutagenesis is carried out.

A

In oligonucleotide-directed mutagenesis, an oligonucleotide containing the desired mutation in the sequence is synthesized. This mutant oligonucleotide is annealed to denatured target DNA template and used to direct DNA synthesis. The result is a double-stranded DNA molecule with a mismatch at the site to be mutated. When transformed into bacterial cells, bacterial repair enzymes will convert the molecule to the mutant form about 50% of the time.

27
Q

Describe how knockout mice are produced and how they are used

A

First, the target gene is cloned. The middle portion of the gene is replaced with a selectable marker, typically the neo gene that confers resistance to the drug G418. This construct is then introduced back into mouse embryonic stem cells and cells with G418 resistance are selected. The surviving cells are screened for cells where the chromosomal copy of the target gene has been replaced with the neo-containing construct by homologous recombination of the flanking sequences. These embryonic stem cells are then injected into mouse blastocyst-stage embryos and these chimeric embryos are transferred to the uterus of a pseudopregnant female mouse. The knockout cells will participate in the formation of many tissues in the mouse fetus, including germ-line cells. The chimeric offspring are interbred to produce offspring that are homozygous for the knockout allele.
The phenotypes of the knockout mice provide information about the function of the gene.

28
Q

How is RNA interference used in the analysis of gene function?

A

RNA interference is one potential reverse genetics approach to analyze gene function, by specifically repressing expression of that gene. Double-stranded RNA may be injected directly into a cell or organism or the cell or organism may be genetically modified to express a double-stranded RNA molecule corresponding to the target gene.

29
Q

___ ____ is the correction of a defective gene by either gene replacement or the addition of a wild-type copy of the gene. For this to work, enough of the cells of the critically affected tissues or
organs must be transformed with the functional copy of the gene to restore normal physiology.

A

gene therapy

30
Q

The introduction to this chapter discusses how gene therapy is used to treat Leber congenital amaurosis (LCA, a type of severe blindness). What characteristics of blindness that affect the retina (such LCA) make it an attractive candidate for treatment by gene therapy?

A

Retinal cells are limited in number and localized to the back of the eye, where they are relatively accessible. Light perception and vision are easily quantified, so that the effects of gene therapy can be measured. Because blindness is a major handicap, even partial success is highly beneficial to the patient. The genes responsible for the disease are known and can be tested to determine where the mutation lies before gene therapy.

31
Q

Will restriction sites for an enzyme that has 4 bp in its restriction site be closer together, farther apart, or similarly spaced, on average, compared with those of an enzyme that has 6 bp in its restriction site? Explain your reasoning.

A

The restriction sites for an enzyme with a 4-bp recognition sequence should be spaced closer together than the sites for an enzyme with a 6-bp recognition sequence. The 4-bp recognition sequence will occur with an average frequency of once every 44 = 256 bp, whereas the 6-bp recognition sequence will occur with an average frequency of once every 46 = 4096 bp.

32
Q

You have discovered a gene in mice that is similar to a gene in yeast. How might you determine whether this gene is essential for development in mice?

A

This gene must first be cloned, possibly by using the yeast gene as a probe to screen a mouse genomic DNA library. The cloned gene is then used to create a knockout mouse strain. A neo gene is inserted into a cloned exon sequence, followed by the tk (thymidine kinase) gene. This construct is then introduced into mouse embryonic stem cells. Selection of G418 resistant and gancyclovir resistant cells will identify transformants where homologous recombination resulted in insertion of the neo gene into the target mouse gene exon. These knockout stem cells are injected into blastocyst stage embryos, which are then transferred to the uterus of a pseudopregnant mouse. The progeny are tested for the presence of the knockout allele. Progeny with the knockout allele are interbred. If the gene is essential for embryonic development, no homozygous knockout mice will be born. The arrested or spontaneously aborted fetuses can then be examined to determine how development has gone awry in fetuses that are homozygous for the knockout allele.