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a mutation is
heritable change in the genetic material
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a mutant is..
an individual, organism, or new genetic character arising or resulting from mutation
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The genotype is...
the genetic makeup of an organism
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The phenotype is...
an organism's physical traits
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Prototroph has what nutritional needs?
only simple nutrional needs
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Auxotrophs require what for nutritional needs?
one or more complex biolmolecules
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Something that is lethal is displayed as death as a _______.
phenotype
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Things that are temperature sensitive are considered to be ________lethal
conditional
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When one purine changes to another purine or
When one pyrimidine changes to another pyrimidine, it is called..
a transition
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When purine changes to pyrimidine or vice versa, it is called..
transversion
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When a codon is changed for one amino acid to another, it is called...
missense
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when a codon change for an amino acid to a stop codon it is called...
nonsense
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What is the polypeptide chain intiation codon?
AUG
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What are the polypeptide chain termination codons?
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What are the four types of reversion?
- Forward mutation
- Reverse mutation
- True reversion
- Second site reversion
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When you go from Wild Type to mutant it is a ______mutant
forward
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When you go from mutant back to Wildtype it is a _____ mutation
reverse
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When you return to original amino acid at mutant site, it is called
true reversion
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when you change at a second site to compensate for the first change it is called...
second site reversion
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What is the stages of shapes in a second site reversion?
- proper shape
- imporper shape
- nearly proper shape
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What is suppression?
Similar to reversion except it occurs on a second gener
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What is a nonsense suppression?
second site mutation that reverts a nonsense mutation (premature stop codon)
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What are the two causes of spontaneous mutations?
- mistakes during replication
- rare tautomers (unusual arrangments of the bases)
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when you have a spontaneous deamination of the amino group from a base pair, the NH2 is replaced by...
an O
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when the spontaneous deamination of cyotsine takes places it turns into...
Uracil
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when the spontaneous deamination of cyotsine takes places it turns into uracil, when it does this, DNA repair enzymes can recognize uracil as an _______ base in DNA and...
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What are the 6 steps of urcail repair by DNA glycosylase
- 1) deamination
- 2) biding of uracil DNA glycosylase
- 3) Excision or Uracil
- 4) Sugar phosphate removed by AP endonuclease and phosphodiesterase
- 5) DNA polymerase
- 6) DNA ligase
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Thymine is a _______ of DNA
normal constiuent
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The fact that thymine is a normal constituent of DNA is a problem for repair enzymes because?
It cannot determine which of the two bases on the two DNA strands is correct
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Methylated cytosine bases tend to create _______ for mutation.
hot spots
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_______ is created as part of the DCM methylation system.
5-methyl cytosine
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5 methyl Uracil is...
Thymine
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Base analgues become incorporated into DNA during...
replication
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5 bromouracil is a _____ analogue
thymine
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What can get incorporated into DNA instead of thymine?
5-bromouracil
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What are five types of potent chemical mutagens?
- alkylating agents
- base analogs
- Acridines
- deaminating agent
- hydroxylating agent
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Frame shift mutations are..
a genetic mutation caused by indels (insertions or deletions) of a number of nucleotides that is not evenly divisible by three from a DNA sequence.
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Proflavin molecules intercalated into a dna double helix causes ________ mutations.
Frame shift
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Proflavin molecules differ from other mutagens because...
it causes basepair-deletions or basepair-insertions and not substitutions.
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UV light induced thymine dimers are formed by...
Two thymines reacting with UV light
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In the center of a thymier dimer is a _____ ring
cyclobutyl
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What are the steps of repair of thymine dimers by photolyase?
- 1) UV irradiation
- 2) photolyase binds to the thymine dimer in DNA
- 3) Cleavage of cross links
- 4) Release of enzyme
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How would nonsense mutations affect the amount of functional protein that is expressed from a gene?
inhibit protein function, particularly if it was not near the end of the coding sequence.
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How would missense mutations affect the amount of functional protein that is expressed from a gene?
It may or may not affect protein function, depending on the nature of the amino acid substitution and whether the substitution is in a critical region of the protein.
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How would up promoter mutations affect the amount of functional protein that is expressed from a gene?
It would increase the amount of functional protein.
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How would mutation that affect splicing affect the amount of functional protein that is expressed from a gene?
It may affect protein function if the alteration in splicing changes an exon in the mRNA that results in a protein with a perturbed structure
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Give two examples of how a mutagen can interfere with DNA replication to cause a mutation.
- A thymine dimer can interfere with DNA replication because DNA polymerase cannot slide past the dimer and add bases to the newly growing strand.
- Alkylating mutagens such as nitrous acid will cause DNA replication to make mistakes in the base pairing
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During mismatch repair, why is it necessary to distinguish between the template strand and the newly made daughter strand?
Mismatch repair is aimed at eliminating mismatches that may have occurred during DNA replication.
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How is the distinction between the template strand and the newly made daughter strans accomplished?
MutH binds to the hemimethylated DNA in a way that allows the mismatch repair system to distinguish which strand is methylated and which is not.
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What is the underlying genetic defect that causes xeroderma pigmentosum?
a defect in one of the genes that encode a polypeptide involved with nucleotide excision repair.
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How can the symptoms of this xeroderma pigmentosum be explained by the genetic defect?
- defective in repairing DNA abnormalities such as thymine dimers and abnormal bases.Therefore, they are very sensitive to environmental agents such as UV light.
- UV light is more likely tocause mutations in these people compared to unaffected individuals. For this reason, people with XP develop pigmentation abnormalities and premalignant lesions and have a high predisposition to skin cancer.
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What are the general steps of cloning?
- Have a vector DNA and a chromosoal DNA
- cut the DNAs with the samerestriction enzyme
- Mix the DNAs together, allo time for sticky ends to basepair
- Add DNA ligase to covalently link a piece of chromosome DNA into the plasmid
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What is a restriction enzyme?
is an enzyme that cuts DNA at specific recognition nucleotide sequences known as restriction sites
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DNA ligase ______ link the DNA backbones in cloning
Covalently
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Unlike gene cloning, PCR can copy DNA without...
the aid of vectors and host cells
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The starting material for PCR includes..
- Tmeplate DNa
- Oligonucleotide Primers
- Deoxynucleoside triphosphates (dNTPs)
- Taq polymerase
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The Template DNA for PCR conatins...
the region that needs to be amplified
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The oligonucleotide primers of PCR are complementary to sequences at the ends of
the DNA fragment to be amplified
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The oligonucleotide primers of PCR are _____ and about 15-20 nucleotides long.
synthetic
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The deoxynucleoside triphosphates (dNTPs) of PCR provide the precursors for...
DNA synthesis
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Taq polymerase is DNA polymerase isolated from the bacterium...
Thermus Aquaticus
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Taq polymerase is a ______ enzyme.
Thermostable
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Taq polymerase is neceassary for PCR because...
PCR involves heating steps that inactivate most other DNA polymerases
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To clone eukaryotic genes without the introns, one can start with a sample of...
mRNA
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To clone eukaryotic genes without the introns, one can start with a sample of mRNA while using what enzyme? why?
- reverse transcriptase
- because it uses RNA as a template to make a complementary strand of DNA
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DNA that in made from RNA is called...
cDNA (complementary DNA)
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What are the steps of the production of cDNA?
- add a poly-dT primer
- add reverse transcriptase and dNTPs to synthesize a cDNA strand
- Add RNaseH to cut up the RNA and generat RNA primers
- Add DNA polymerase ans DNA ligase to synthesize the second DNA strand
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To transform ligated plasmids into E. Coli, what should you do?
- Mix DNA with many ecoli cells that have been treated with agents to make them permeable to DNA
- Plate cells on Agar medium with antibiotic
- all cells in a colony are genetically identical
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What are three examples of cloning vectors?
- pBR322 (stone tools)
- pBluescript
- pET (for protein expression)
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Blue/White selection is based on...
β-galactosidase and a dye called 5-bromo-4-chloro-3-indoyl-b-D-galactoside or X-Gal.
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pBluescript is..
a commercially available phagemid containing several useful sequences for use in cloning with bacteriophage.
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When lactose is present in the environment, The binding of the inducer allolactose prevents the .....
lac repressor from binding to the operator site
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What are the steps with cloning with pUC plasmids?
- Transform JM strain of E. coli: contains F plasmid with part of β-Galactosidase
- Plate transformants on agar plates with IPTG and X-Gal
- Blue colonies contain intact plasmid
- White colonies have inactivated the coding segment for the α fragment
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You can determin the sequence of DNA by which method?
Dideoxy chain termination method
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What is the dideoxy method?
- DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5’–P of one and the 3’–OH of the other
- Nucleotides missing that 3’–OH can be synthesized
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What is chain termination?
When a growing DNA strand canno longer grow because a dideoxynucleotide is added to it.
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the dideoxy sequencing method is automated using...
a laser and fluorescent detector
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The fragments in the dideoxy sequencing method are seperated by...
Gel electrophoresis
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The detector reads the level of fluorescence at ____wavelengths
four
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What is one reason to clone genes?
to express recombinant proteins
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What are the steps to clone and express HGH?
- Obtain the amino acid sequence
- create a cDNA library for HGH
- Determine the DNA sequence of your choice
- Prepare an expression plasmid with your sequence of HGH
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IGF-1 stands for...
Insulin-like Growth Factor 1 (easier to measure than HGH)
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