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BMSC 210 > Chapter 12 > Flashcards

Chapter 12 Flashcards

1
Q

Genetic engineering

A

the use of in vitro techniques to alter genetic material in the lab

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2
Q

First gene clone and purified from pigs

A

insulin

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3
Q

Recombinant DNA technology

A

the artificial recombination of DNA from two organisms

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4
Q

Restriction Endonuclease Enzyme

A
  • recognition specific DNA sequences and cut DNA

- essential for in vitro DNA manipulation and gene cloning

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5
Q

Methylation

A

cells must protect themselves from inadvertent destruction

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6
Q

Type II

A
  • cleave DNA within recognition sequence

- most useful for specific DNA manipulation

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7
Q

Type I

A
  • first discovered/purified
  • cuts DNA at random far from recognition sequence
  • little practical value
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8
Q

Type III

A
  • large
  • cleaves outside of recognition
  • rarely used
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9
Q

How do structures of cleaved products differ?

A

3’ or 5’ overhang or blunt ends

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10
Q

recognition sequence are usually….

A

4-8 bp long

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11
Q

how are the 3 letters in REs designated?

A
  • first letter is genus RE isolated
  • next two represent species of RE isolated from
  • Roman numeral indicated order of discovery
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12
Q

Escherichia coli enzyme designation

A

EcoRI, EcRV

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13
Q

Escherichia coli recognition sequence

A

I: G|AATTC
V: CTTAA|G

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14
Q

DNA fragments produced by EcoRI

A

5’ overhangs

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15
Q

DNA fragments produced by Pstl

A

3’ overhangs

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16
Q

DNA fragments produced by Smal

A

blunt ends

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17
Q

The nucleic acid sequence where EcoRI cuts

A

GAATTC

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18
Q

Molecules with complimentary sticky ends can easily….

A

anneal or form H bonds

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19
Q

Ligation

A

by DNA ligase can rejoin two sugar phosphate backbones of DNA through covalent bonding

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20
Q

After modification, DNA can no longer….

A

be cut by a RE

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21
Q

3 main steps of gene cloning

A
  1. isolation and fragmentation of source DNA
  2. insertion of DNA fragments into cloning vector
  3. intro of cloned DNA into host organism
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22
Q

plasmids

A

natural vectors and have useful properties of cloning vectors

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23
Q

why are plasmids useful?

A
  • small
  • independent origin of replication
  • multiple copy # so multiple copies of cloned genes
  • presence of selectable markers
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24
Q

vector transfer to host can be accomplished by

A

chemical transformation of host cell, sometimes conjugation or transduction, or electroporation

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25
Q

essential features of plasmid pUC19

A
  • ampicillin resistance, polylinker with multiple restriction enzyme cut sites,
  • lacZ is fully functional even with presence of polylinker
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26
Q

pUC19 size

A
  • small

- 2686 bp

27
Q

enzyme used for cloning

A
  • restriction endonuclease
  • DNA ligase
  • reverse transcriptase
  • DNA polymerase
28
Q

DNA ligase

A

catalyzes the going of two strands of DNA between 5’ and 3’ adjacent nucleotides with either cohesive or blunt ends

29
Q

reverse transcription

A

converts RNA into DNA

30
Q

DNA polymerase

A

mostly used for 5’3’ polymerizing activity

- also have 3’5’ and 5’3’ exonuclease activity

31
Q

blue colonies

A

do NOT have vector with foreign DNA inserted

32
Q

white colonies

A

have foreign DNA inserted

33
Q

genomic library

A

nearly/complete copy of organisms genome contained as recombinant DNA plasmids/ phages

34
Q

To clone only expressed genes, libraries can be constructed using….

A

organisms mRNA

35
Q

how is mRNA cloned?

A

not directly.. used as template for retroviral reverse transcriptase

36
Q

common host strains

A

E. coli, Bacillus subtilis, saccharmoyces cervisiae

37
Q

ideal hosts should be….

A
  • rapid growth
  • non pathogenic
  • incorporates DNA
  • stable
  • allows replication of vector
38
Q

E. coli

A

pro: well developed, main strains, well know
cons: pathogenic, traps proteins

39
Q

bacillus subtilis

A

pros: easily transformed, nonpathogenic, secreted proteins
cons: unstable, less developed

40
Q

saccharomyces cerevisiae

A

pros: well developed, nonpathogenic, processes mRNA, easy to grow
cons: unstable, doesn’t replicate bacterial plasmids

41
Q

Agarose Gel Electrophoresis

A

separates DNA by charge/size

42
Q

Agarose gels can be stain with ______ and DNA is visualized under _______.

A

ethidium bromide

UV light

43
Q

the same DNA cut with different restriction enzymes will have _____ banding patterns on an agarose gel.

A

different

44
Q

RE map

A

a map of location of restriction enzyme cuts on a segment of DNA

45
Q

PCR

A

Polymerase Chain Reaction

  • DNA replication in a test tube
  • rapid amplification in number of copies of specific DNA
46
Q

applications of PCR

A
  • determine DNA from a particular region
  • cloning specific fragments
  • identifying DNA source (crime)
  • paternity tests
  • ancient DNA compared to modern
  • ascertaining difficult to culture organisms
47
Q

steps in PCR amplification

A
  1. denatured by heating
  2. synthetic DNA is added
  3. DNA polymerase is added (Taq poly/ Pfu poly)
  4. heat & cool
48
Q

a variation of PCR

A

reverse transcriptase PCR

  1. add primer and reverse transcriptase
  2. form single stranded cDNA
  3. add RNase H
  4. add primer specific to 5’ end + taq = double stranded cDNA
49
Q

nucleic acid hybridization

A

base pairing of single stranded DNA/RNA from 2 different sources to give hybrid double helix

50
Q

nucleic acid probe

A

segments of single-stranded DNA that is used for hybridization
*ss DNA complementary to the sequence of gene of interest

51
Q

how are nucleic acid probes created?

A

cloning, synthesis, denaturing, or fragment of DNA

52
Q

molecular beacon

A

radioactive, fluorescent dye

53
Q

synthesized DNA is used for…

A

primers and probes, and in site-directed mutagenesis

54
Q

FISH

A

Fluorescent In Situ Hybridization

-fluorescent probes attach to Olig.

55
Q

Southern blot

A
  1. labelled DNA probes is used to find complementary sequence by hybrid. with radioactive probe
  2. position of hybrid band is noted using X-ray/radiography or fluorescent
  3. only some DNA fragments hybridize.
56
Q

Northern blot

A
  • RNA is in the gel
  • radioactive probe to specific gene
  • same blot probed with 5S RNA as a control
57
Q

how to detect clones containing correct DNA inserts

A
  • must establish that cloning procedures worked
  • initial selection– antibiotic resistance; white-blue screening
  • if cells express foreign gene, expression might be detected
  • look for specific DNA id the gene is not expressed.
58
Q

reporter genes

A

encodes proteins easy to visualize

i.e. lacZ, luciferase, green fluorescents

59
Q

gene fusions

A

promotors or coding sequences of gene of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions

60
Q

site-directed mutagenesis

A

performed in vitro and introduces mutations at a precise location

61
Q

cassete mutagenesis/knockout mutations

A

DNA fragment can be cut, excised and replaced by synthetic DNA fragments

62
Q

gene disruption/insertional inactivation

A

cassettes (i.e. antibiotic resistance genes) are inserted into a gene and disrupting its function

63
Q

knockout mutation

A

total loss of gene function when disrupted gene recombines into chromosome

BMSC 210 (4 decks)

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