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Molecular Biology > Chapter 4: Molecular Cloning Methods > Flashcards

Chapter 4: Molecular Cloning Methods Flashcards

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)

How well did you know this?
1
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16
Q

produced by Haemophilus influenzae

blunt-end cutter

GTPy–PuAC

CAPu–PyTG

A

HindII

How well did you know this?
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2
3
4
5
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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)

A

bluescript II

20
Q

creates phosphodiester bonds to link DNA

A

DNA ligase

21
Q

7 types of vectors

A
  1. bacteriophages (e.g. Lambda phage)
  2. Charon 4
  3. pUC
  4. cosmid (large plasmids)
  5. M13 phage
  6. Phagemids
  7. bluescript
22
Q

an identical copy of a gene, cell, or organism

22
Q

aka R-M system: consists of restriction enzymes and methylases

A

restriction modification system (aka?)

24
Q

restriction endonucleases

A

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

25
Q

clone

A

an identical copy of a gene, cell, or organism

27
Q

Cohen & Boyer

A

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
Q

cloning a gene into a vector already containing a gene that can be readily detected

i.e. green fluorescent protein (GFP) or oligohistidine

A

fusion protein (i.e.)

28
Q

bluescript II

A

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

29
Q

HindIII

31
Q

PCR

A

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
Q

NotI

A

GC–GGCCGC

32
Q

Stanley Cohen, 1973

A

first cloning experiment (name, date)

33
Q

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

36
Q

DNA ligase

A

creates phosphodiester bonds to link DNA

37
Q

fusion protein (i.e.)

A

cloning a gene into a vector already containing a gene that can be readily detected

i.e. green fluorescent protein (GFP) or oligohistidine

38
Q

sequences containing a 2-fold symmetry that read the same forward or backward

i.e. EcoRI site

A

palindromes (i.e.)

40
Q

reverse transcriptase

A

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

41
Q

RNase H

A

destroys RNA; used in cDNA to prevent addition of complementary strand of DNA by DNA polymerase

43
Q

palindromes (i.e.)

A

sequences containing a 2-fold symmetry that read the same forward or backward

i.e. EcoRI site

44
Q

G–GATCC

45
Q

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
A

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

46
Q

destroys RNA; used in cDNA to prevent addition of complementary strand of DNA by DNA polymerase

47
Q

A–AGCTT

48
Q

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

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

CCC–GGG

C–CCGGG

A

Isochizomers (e.g.)

49
Q
  1. bacteriophages (e.g. Lambda phage)
  2. Charon 4
  3. pUC
  4. cosmid (large plasmids)
  5. M13 phage
  6. Phagemids
  7. bluescript
A

7 types of vectors

51
Q

restriction modification system (aka?)

A

aka R-M system: consists of restriction enzymes and methylases

52
Q

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

53
Q

bidirectional cloning (i.e.)

A

cloning a piece of DNA that’s been cut with 2 different enzymes

i.e. EcoRI & PstI

54
Q

BamHI

55
Q

cloning a piece of DNA that’s been cut with 2 different enzymes

i.e. EcoRI & PstI

A

bidirectional cloning (i.e.)

56
Q

first cloning experiment (name, date)

A

Stanley Cohen, 1973

Molecular Biology (13 decks)

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