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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 organism

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4
Q

Restriction Endonuclease Enzymes

A
  • recognize 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 their own DNA 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 sequences
  • 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 sequences are usually….

A

4-8bp long

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11
Q

How are the 3 letters in REs designated?

A
  • first letter is genus RE isolated from
  • next two represent species RE isolated from
  • roman numeral indicated over of discovery in that species
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12
Q

Escherichia coli enzyme designation

A

EcoRI, EcoRV

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13
Q

Escherichia coli recognition sequence

A

I: G|AATTC
V: GAT|ATC

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14
Q

DNA fragments produced by EcoRI

A

5’ overhangs

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15
Q

DNA fragments produced by PstI

A

3’ overhangs

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16
Q

DNA fragments produced by SmaI

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 complementary 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 binding

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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
    - insertion of DNA fragment into cloning vector
    - introduction of cloned DNA into host organism
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22
Q

plasmids

A

natural vectors and have useful properties are 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 gene per cell
  • 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

Enzymes used for cloning

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

DNA ligase

A

catalyze joining of two strands between 5’ phosphate and 3’ OH of adjacent nucleotides

29
Q

Reverse transcriptase

A

converts RNA into DNA

30
Q

DNA polymerase

A

Mostly used for 5’3’ polymerizing activity; may 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

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

34
Q

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

A

the organisms’ mRNA

35
Q

How is mRNA cloned

A

It cannot be cloned directly and is used as a template for retroviral reverse transcriptase

36
Q

common host strains

A

E coli, bacillus subtitles, saccharomyces cerevisiae

37
Q

Ideal hosts should be…

A
  • rapid growth in inexpensive medium
  • nonpathogenic
  • capable of incorporating DNA
  • genetically stable in culture
  • equipped with appropriate enzymes to allow replication of the vector
38
Q

E coli

A
  • well developed genetics
  • many strains
  • best known bacteria
  • potentially pathogenic and periplasm traps proteins
39
Q

bacillus subtilis

A
  • easily transformed
  • nonpathogenic
  • secretes proteins
  • endospore formation simplifies culture
  • genetically unstable and genetics less developed
40
Q

saccharomyces cerevisiae

A
  • well-developed genetics
  • nonpathogenic
  • can process mRNA and proteins
  • easy to grow
  • plasmids unstable and will not replicate most bacterial plasmids
41
Q

Agarose Gel Electrophoresis

A
  • separates DNA molecules based on size

- uses electrical field to separate charged molecules; nucleic acids migrate towards positive electrode

42
Q

Agarose gels can be stained with ___________ and DNA is visualized under _________.

A

ethidium bromide, UV light

43
Q

The same DNA cute with different restriction enzymes will have ___________ banding patterns on an agarose gel.

A

different

44
Q

RE map

A

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

45
Q

PCR

A

DNA replication in a test tube - DNA amplification

-rapid amplification in the # of copies of specific DNA sequences for further analysis

46
Q

Applications of PCR

A
  • determing DNA sequences
  • cloning a specific fragment
  • identifying DNA source from a crime scene
  • paternity determination
  • analysis of ancient DNA
  • organisms presence in a specimen
47
Q

Steps in PCR amplification

A
  • DNA denatured by heat
  • add DNA pol
  • Ad Taq pol
  • heat and cool many many many times in similar cycles
48
Q

A variation of PCR

A

reverse transcriptase PCR

49
Q

Nucleic Acid Hybridization

A

Base pairing of single strands of DNA or RNA from 2 different sources to give a hybrid double helix

50
Q

nucleic acid probe

A

segment of single-stranded DNA used in hybridization and has a predetermined identity

51
Q

How are nucleic acid probes created?

A

By cloning, synthesis, or denaturing a fragment of DNA

52
Q

molecular beacon

A

label probes

53
Q

Synthesized DNA is used for..

A

primers and probes, and in site directed mutagenesis

54
Q

FISH

A

Fluorescent In Situ Hybridization; uses fluorescent probe attached to oligonucleotide

55
Q

Southern Blot

A

a hybridization procedure where DNA is in the gel and probe is RNA or DNA

56
Q

Northern Blot

A

RNA is in the gel; Radioactive probe to a specific gene to total RNA which is run on a gel

57
Q

How to detect clones containing correct DNA inserts

A
  • establish if cloning procedure worked
  • antibiotic resistance/blue-white screening
  • if cells express the foreign gene
  • look for specific DNA is the gene is not expressed
58
Q

reporter genes

A
  • encode proteins that are easy to visually detect and assay

- lacZ, luciferase, green fluorescent protein

59
Q

gene fusions

A

promoters or coding sequences of genes 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
-can be used to assess activity of specific amino acids in a protein

61
Q

cassete mutagenesis/knockout mutations

A

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

62
Q

gene disruption/ insertional inactivation

A

When cassettes are inserted into a gene disrupting its function
-may cause knockout mutations

63
Q

knockout mutations

A

total loss of gene function; great for studying what genes do

BMSC 210 (15 decks)

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