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Molecular Biology > Chapter 4: Molecular Cloning Methods > Flashcards

Chapter 4: Molecular Cloning Methods Flashcards

(56 cards)

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1
Q

EcoRI

A

sticky-end cutter

5’ G–AATTC 3’

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1
Q

protein enzymes produced in bacteria (especially E. coli) that cut up DNA

A

restriction endonucleases

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1
Q

utilizes heat-stable DNA polymerases to synthesize new DNA strands; performed by the heat cycler

heat DNA/denature –> prime DNA –> polymerize new DNA strands –> cool/anneal –> repeat

A

PCR

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2
Q

Isochizomers (e.g.)

A

restriction enzymes that recognize the same nucleotide sequence but cut at different sites

e.g. SmaI (blunt-end cutters) & XmaI

CCC–GGG

C–CCGGG

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2
Q

luciferin

A

expressed in fireflies w/ luciferase gene, causing fluorescence in transformed pigs and tobacco plants as well

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3
Q

small, circular DNAs that are independent of the bacterial chromosomes

A

plasmid

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4
Q

primer

A

short sequences of DNA/RNA that bind specific sequences to initiate DNA synthesis

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4
Q

complementary DNA or copy DNA library, generated from all RNA’s expressed in the cell

requires the use of RNA as the starting material

each cDNA represents a gene clone from a particular cell

A

cDNA cloning

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4
Q

used the sticky-end cut of EcoRI to cut 2 plasmids (pSC101 & RSF1010) and place the recombinant DNA into bacteria; confirmed with tetracycline and streptomycin (sulfonamide resistance)

A

Cohen & Boyer

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5
Q

methylase

A

enzyme that place methyl groups on nucleotides and “protect” the DNA from restriction

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6
Q

partially methylated DNA strand (aka)

A

hemi-DNA; protected against cleavage

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7
Q

sticky-end cutter

5’ G–AATTC 3’

A

EcoRI

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8
Q

hemi-DNA; protected against cleavage

A

partially methylated DNA strand (aka)

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9
Q

HindII

A

produced by Haemophilus influenzae

blunt-end cutter

GTPy–PuAC

CAPu–PyTG

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9
Q

enzyme that removes the 5’ phosphates used by DNA ligase for ligating (helps prevent reclosing of plasmid)

A

alkaline phosphatase

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11
Q

plasmid

A

small, circular DNAs that are independent of the bacterial chromosomes

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11
Q

synthesizes DNA from RNA; requires an Oligo (dT) primer when used to make cDNA

A

reverse transcriptase

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12
Q

vector (must contain 3 things) (i.e.)

A

plasmids expressed in bacteria that allow for the replication of the DNA

  1. origin of replication
  2. antibiotic resistance gene(s)
  3. multiple restriction sites
    i. e. pBR322
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14
Q

cDNA cloning

A

complementary DNA or copy DNA library, generated from all RNA’s expressed in the cell

requires the use of RNA as the starting material

each cDNA represents a gene clone from a particular cell

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15
Q

alkaline phosphatase

A

enzyme that removes the 5’ phosphates used by DNA ligase for ligating (helps prevent reclosing of plasmid)

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16
Q

produced by Haemophilus influenzae

blunt-end cutter

GTPy–PuAC

CAPu–PyTG

A

HindII

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17
Q

expressed in fireflies w/ luciferase gene, causing fluorescence in transformed pigs and tobacco plants as well

A

luciferin

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18
Q

GC–GGCCGC

19
Q

contains lacZ gene, origin of replication, Amp gene, lacI (B-galactosidase)

Study These Flashcards
A

bluescript II

20
creates phosphodiester bonds to link DNA
DNA ligase
21
7 types of vectors
1. bacteriophages (e.g. Lambda phage) 2. Charon 4 3. pUC 4. cosmid (large plasmids) 5. M13 phage 6. Phagemids 7. bluescript
22
an identical copy of a gene, cell, or organism
clone
22
aka R-M system: consists of restriction enzymes and methylases
restriction modification system (aka?)
24
restriction endonucleases
protein enzymes produced in bacteria (especially *E. coli*) that cut up DNA
25
clone
an identical copy of a gene, cell, or organism
27
Cohen & Boyer
used the sticky-end cut of EcoRI to cut 2 plasmids (pSC101 & RSF1010) and place the recombinant DNA into bacteria; confirmed with tetracycline and streptomycin (sulfonamide resistance)
27
cloning a gene into a vector already containing a gene that can be readily detected i.e. green fluorescent protein (GFP) or oligohistidine
fusion protein (i.e.)
28
bluescript II
contains lacZ gene, origin of replication, Amp gene, lacI (B-galactosidase)
29
HindIII
A--AGCTT
31
PCR
utilizes heat-stable DNA polymerases to synthesize new DNA strands; performed by the heat cycler heat DNA/denature --\> prime DNA --\> polymerize new DNA strands --\> cool/anneal --\> repeat
32
NotI
GC--GGCCGC
32
Stanley Cohen, 1973
first cloning experiment (name, date)
33
short sequences of DNA/RNA that bind specific sequences to initiate DNA synthesis
primer
36
DNA ligase
creates phosphodiester bonds to link DNA
37
fusion protein (i.e.)
cloning a gene into a vector already containing a gene that can be readily detected i.e. green fluorescent protein (GFP) or oligohistidine
38
sequences containing a 2-fold symmetry that read the same forward or backward i.e. EcoRI site
palindromes (i.e.)
40
reverse transcriptase
synthesizes DNA from RNA; requires an Oligo (dT) primer when used to make cDNA
41
RNase H
destroys RNA; used in cDNA to prevent addition of complementary strand of DNA by DNA polymerase
43
palindromes (i.e.)
sequences containing a 2-fold symmetry that read the same forward or backward i.e. EcoRI site
44
G--GATCC
BamHI
45
plasmids expressed in bacteria that allow for the replication of the DNA 1. origin of replication 2. antibiotic resistance gene(s) 3. multiple restriction sites i. e. pBR322
vector (must contain 3 things) (i.e.)
46
destroys RNA; used in cDNA to prevent addition of complementary strand of DNA by DNA polymerase
RNase H
47
A--AGCTT
HindIII
48
restriction enzymes that recognize the same nucleotide sequence but cut at different sites e.g. SmaI (blunt-end cutters) & XmaI CCC--GGG C--CCGGG
Isochizomers (e.g.)
49
1. bacteriophages (e.g. Lambda phage) 2. Charon 4 3. pUC 4. cosmid (large plasmids) 5. M13 phage 6. Phagemids 7. bluescript
7 types of vectors
51
restriction modification system (aka?)
aka R-M system: consists of restriction enzymes and methylases
52
enzyme that place methyl groups on nucleotides and "protect" the DNA from restriction
methylase
53
bidirectional cloning (i.e.)
cloning a piece of DNA that's been cut with 2 different enzymes i.e. EcoRI & PstI
54
BamHI
G--GATCC
55
cloning a piece of DNA that's been cut with 2 different enzymes i.e. EcoRI & PstI
bidirectional cloning (i.e.)
56
first cloning experiment (name, date)
Stanley Cohen, 1973

Molecular Biology (13 decks)

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