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59 Cards in this Set
- Front
- Back
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The most commonly used enzymes for DNA manipulation, maintenance, storage, repair, replication and expression are ___, ___, ___, ___, ___, ___, ___ and these enzymes are the tools of recombinant DNA technology
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DNA polymerase (DNA-->DNA), DNA ligase (seals), reverse transcriptase (RNA--->DNA), ribonuclease, deoxyribonuclease, the function of primase, and restriction enzymes
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It was observed that bacteriophages grown on one strand of E. coli could be grown with high efficiency on the ____ strain but not on ____ strains of E. coli
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same
distantly-related |
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Any phage that happened to survive after infecting a distantly-related strain could be easily propagated on _____ strain but not on _____
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that
original E coli. |
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Scientists concluded that the growth on the phage was partially "____" to the strain that served as the most recent host
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restricted
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DNA-cutting enzymes called ______ _______ are responsible for bacteriophage restriction
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restriction enzymes
(restriction endonucleases) |
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Restriction Enzymes generally recognize __, __, __, __ nucleotides
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4, 6, 8, 12
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Many Restriction Enzymes bind to DNA and digest it at positions where adenines are methylated (Type ____)
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1 & 3
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Other Restriction Enzymes are digested at specific DNA sequences, type ___, which are more useful
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2
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Restriction Enzymes are often _____ and cut at ____ sites
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dimers
palindromic |
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Example of palindromic
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5'-GAATTC-3'
3'-CTTAAG-5' -reads the same frontwards on one strand and backwards on the complementary strand |
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Four nucleotides occur at random every ___ nucleotides, so restriction enzymes that recognize ____ nucleotides are generally used which occur at random every 4096 nucleotides
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256
6 (based on the fact that 50% is GC base pairs and 50% is AT bp) |
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Reaction enzymes are not always _____-specific and if the reaction is allowed to proceed for too long, or in an incorrect ____ then digestion at _____ can occur; this is known as _____
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site
buffer non-specific sites star activity |
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Restriction enzymes are used by ____ as a defense against bacteriophages
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bacteria
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Phages could not infect ___ strains of E. coli because that strain strain had restriction enzymes that digested any ______
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new
unrecognized |
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E. coli has ~4.6 million bp and it would be impossible to prevent ________
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its own restriction enzymes from cutting its own DNA
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The solution to this problem is to make use of DNA ____ which are used to determine the original vs. new strand in DNA replication
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methylases
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These methylases will methylate the entire genome within a few minutes of replication at specific _____
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adenines
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Usually, the restriction endonucleases can cleave only DNA that is completely _____
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unmethylated
BOTH STRANDS |
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Upon replication of the methylated DNA, the host methylates 1 strand of DNA, called ____, via methylase enzymes unique to that _____
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hemi-methylation
strain of bacteria |
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Hemi-methylated DNA (is/is not) cleaved by restriction endonucleases
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is not
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To survive, a cell can't contain restriction endonuclease unless is also contains _____ that modifies the specific sequence recognized by the restriction endonuclease
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methylase
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A- unmethylated DNA
B- methylated DNA |
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A phage can inject its DNA into E. coli, but ______ because of the E. coli's restriction enzymes
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the DNA is degraded because it is not methylated
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The only way a phage can safely reproduce in an E. coli cell is if its DNA was first _____; the phage is now said to be "_____" to the strain
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methylated
restricted |
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Each strain of bacteria contains different ___ and ___ enzymes that specifically recognize different DNA sequences
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restriction and methylation
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Restriction enzymes are named for their ____ organism, with the ___ and ___ and the last part of the name is the ____
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source
genus species strain EX: EcoRI (Escherichia coli strain R) |
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Restriction enzymes cutting DNA at specific DNA sequences
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Digestion within the recognized DNA sequence may occur ______ generating blunt cleavage termini
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both strands in the center of the recognized sequence
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Or it can occur at _________ generating termini with one strand or the other having an "overhang"
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staggered positions on each strand
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What enzyme cuts at C^CGG?
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HPA2
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Restriction enzymes that cut in the exact same position and manner are called _____
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isoschizomers
SphI and BbulI both cutting CGTAC^G |
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Restriction enzymes that cut the same sequence of DNA but cut in a different fashion are called ______
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neoschizomers
Tail cuts ACGT^ Maell cuts A^CGT |
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Enzymes have specific ____ requirements and to put two different r. enzymes together you should check a ____ table
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buffer
buffer |
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Some r. enzymes require BSA to efficiently work because _____
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it provides a protein rich environment
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Most enzymes need flanking bases placed ____ to bind to the DNA and digest it
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next to the cut site
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Most enzymes cannot perform without an _____ of at least 2-6 xtra bases at the ___' end (usually a GC)
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overhang
5 |
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Restriction enzymes can be used to provide information about a piece of ____ since restriction enzymes cut at specific sites
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DNA sequence
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Restriction enzymes also provide a method to distinguish or identify different DNA molecules of the same ___ but with different ___ without actually sequencing them
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length
sequences |
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In _____ a map is generating of digestion sites across a piece of DNA
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restriction mapping
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Restriction mapping can be used to determine whether an individual is ____ or ____ at a specific locus
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homozygous or heterozygous
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Restriction enzymes have been linked to _____ that recognize DNA and these _____ nucleases may one day be used to repair human point mutations
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zinc finger
zinc finger |
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The fragments that result after cleavage with restriction endonucleases are commonly referred to as ________
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restriction fragments
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# of cuts = ______
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# of pieces - 1
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DNA polymerase is responsible for ______ during synthesis in the _____ direction and making repairs upon DNA damage
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copying DNA
5'-->3' |
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DNA polymerase can also be used to fill ____ after a restriction enzyme digestion to produce blunt ends
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sticky ends
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This allows for ______ regardless of sequence
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two different restriction enzyme digestions to be joined together
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Some DNA polyermases possess _____ directionality and digest downstream DNA or RNA, remove primers, or repair damaged DNA
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5'--->3'
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DNA polymerases with 3'--->5' directionality only ________
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proofread and repair misincorporated bases (improper)
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The Klenow fragment is DNA polymerase subjected to _____; the _____ domain is cleaved; leaving only the proofreading and polymerizing domains
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the protease subtilisin
5'-->3' |
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The Klenow fragment is often used instead of DNA polymerase 1 to _____
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fill in sticky ends, copy ssDNA, or prepare probes
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____ DNA Polymerase's 3'--->5' exonuclease activity is used to chew back ______
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T4
3' overhangs |
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With a 5' overhand "sticky" end you can use ____ to fill in the end to make it blunt
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any polymerase (with 5'-->3' activity)
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To fill in a 3' sticky end you need _____ to yield a blunt ended fragment
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a polymerase with 3'--->5' activity
T4 RNA polymerase |
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DNA ____ glues two fragments of DNA together that have been cleaved
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ligase
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DNA ligase uses ____ to catalyze a covalent intermediate with DNA's phosphate backbone which will then join with the nearby phosphate from another DNA strand
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ATP
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For ligase to function is must have two pieces of DNA that contain _________
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blunt ends or overhangs that are complementary to one another
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DNA linked together must be digested with the same _____ so that the sequences can line up and come together
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restriction enzyme
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This technique is useful for creating ____ as researchers will use 2 different restriction enzymes to ensure that the DNA and ___ fit together perfectly and the direction of the insert can be guaranteed
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plasmids
plasmid |
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____ ligations are better than ____ because the base pairing increases the chances of the two pieces of DNA finding each other in solution
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overhang
blunt end |
