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Biochem 4 > Recombinant DNA > Flashcards

Recombinant DNA Flashcards

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1
Q

name the 4 important achievements leading to modern molecular biology

A
  1. Restriction endonucleases(enzymes)
  2. Cloning of DNA
  3. Creation of synthetic probes
  4. PCR
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2
Q

what do restriction endonucleases do?

A
  • cleave very specific DNA sequences

- usually recognize sequences that are short (4-8 base pairs)

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3
Q

what are the short DNA sequences recognized by restriction enzymes called?

A

palindromes (also called restriction sequences)

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4
Q

how would you identify a palindrome?

A

-it is read the exact same (identical, not base pairs) from 5’ to 3’ direction on both strands

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5
Q

what are the two types of ends that can be formed when a restriction enzyme cleaves a DNA sequence?

A
  • sticky ends

- blunt ends

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6
Q

which end is preferable for ligation? why?

A

sticky end!

-helps orient the DNA you want to ligate

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7
Q

how are restriction enzymes named?

A

-for the organism they were derived from

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8
Q

what is a restriction site?

A
  • DNA sequence that can be cleaved by a restriction enzyme

- same thing as a palidrome

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9
Q

restriction enzymes that recognize LARGER sequences cut more or less frequently than the ones that recognize smaller sequences

A

-enzymes that recognize larger sequences cut less frequently

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10
Q

in general, restriction enzymes are…

A

tools that cut, paste, and analyze DNA

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11
Q

why is it important to know if the restriction enzyme recognizes larger or smaller sequences?

A

-if you are preparing DNA for a gel, you need to know how many fragments you are going to end up with.

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12
Q

Fragments of DNA can be “pasted” together to make a hybrid molecule known as…

A

-recombinant DNA

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13
Q

what enzyme creates the phosphodiester bonds that connect the sequences of DNA in the recombinant DNA?

A

DNA ligase

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14
Q

what are the two ways you can get recombinant DNA?

A
  1. generate it from PCR

2. get it straight from an organism

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15
Q

in the simplest sense, DNA cloning involves inserting a _________ into a ________

A

-inserting a restriction fragment (recombinant DNA) into a cloning vector

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16
Q

when is DNA considered “cloned”

A

as soon as it is inserted into the vector

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17
Q

what is a vector?

A
  • molecules of DNA that can accept fragments of foreign DNA

- usually circular

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18
Q

what are the 3 things a vector must have?

A
  1. must be capable of replication in the cell (organism)
  2. must have at least one restriction site for foreign DNA
  3. must carry at least one gene for selection
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19
Q

what is the most common type of gene for selection used in a vector?

A

antibiotic resistance

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20
Q

why would you use two different restriction enzymes to cut the vector?

A

-to help the orientation of the inserted DNA

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21
Q

what is the most common vector? second most common?

A
  1. prokaryotic plasmids

2. yeast plasmids

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22
Q

what are the two different DNA libraries?

A
  1. Genomic DNA libraries

2. cDNA libraries

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23
Q

the collection of Genomimc DNA libraries contains what?

A

-all sequences in the genome, including coding regions, introns, promoters, etc.

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24
Q

how is the genomimc DNA library made?

A

-ENTIRE genome is chopped up by restriction enzymes, cloned into vectors, and used to transform bacteria

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25
Q

what is cDNA library also known as?

A

-complementary DNA library

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26
Q

how is cDNA generated?

A
  • using isolated mRNA from a particular cell or tissue type
  • mRNA is reverse transcribed and the second strand is synthesized
  • it is then ligated into a vector, used to transform bacteria, and 1000s of clones are collected
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27
Q

what does cDNA library contain?

A

-sequences representing all mRNAs present in the cell or tissue type at the time the mRNA was collected

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28
Q

how much of the genome is represented with genomic DNA libraries? cDNA libraries?

A

genomic-representative of the entire genome

cDNA- only what was collected/present at that time

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29
Q

DNA from a cDNA library can be cloned into an expression vector for production of…

A

proteins!

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30
Q

what does the bacterial expression vector need to transform expression bacteria strains?

A

-promoter, shine-dalgarno sequence, and cDNA

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31
Q

why would you chose to use a yeast vector instead of a bacterial vector?

A

-when you want to express a protein that is integrated into the membrane

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32
Q

what is DNA sequencing used for?

A

to determine the exact sequence of a clones or PCR amplified stretch of DNA

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33
Q

what is used in DNA sequencing to stop DNA elongation? why?

A
  • DIdeoxyribonucleotides
  • ddCTP, ddATP, ddGTP, ddTTP
  • they lack a free OH group, so elongation cannot continue
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34
Q

what are probes used for in general?

A

used to identify DNA fragments

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35
Q

what is a probe?

A

-single standed DNA molecule, labeled using radioactivity (or any label) that can be hybridized with a single stranded DNA molecule that is complementary

36
Q

what is hybridization?

A
  • target DNA is made ssDNA
  • it is immobilized so it cannot reanneal
  • then bathe it in probe
  • probe binds to target
37
Q

how is the target DNA immoblized?

A
  • nitrocellulose membrane has charges associated with it that bind to the DNA
  • it is hydrophobic in nature
38
Q

what are small probes?

A

COMPLETELY CHEMICALLY synthesized oligonucleotides

-2-30 bp

39
Q

what do small probes do?

A
  • they are VERY specific

- can identify a single base mutation in a sequence

40
Q

what are large probes?

A

-made by one of several molecular BIOLOGY techniques (reverse transcriptase, PCR)

41
Q

what do large probes do?

A
  • LESS specific

- can be used to identify similar genes in different organisms OR the same gene in different individuals

42
Q

small probes are more________ and large probes are more_______

A

small probes are more diagnostic and large probes are more comparative

43
Q

Southern blotting is the analysis of_______

A

DNA

44
Q

Northern blotting is the analysis of_______

A

RNA

45
Q

Western blotting is the analysis of______

A

Protein

46
Q

what kind of sequences does Northern blotting detect?

A

-only EXPRESSED sequences

47
Q

which two blotting techniques are quantitative and qualitative?

A
  1. Northern: measures what genes are expressed and how much they are expressed
  2. Western: measures what proteins are expressed and how much they are expressed
48
Q

in Western blotting, the probe is a ______ and sees the protein as a _______

A
  • the probe is an antibody

- the protein is an antigen

49
Q

what is a polymorphism?

A

-genetic variation in non-coding region with no disease associated with them

50
Q

what happens when there are genetic variations in intervening sequences?

A
  • not much

- they are generally not harmful since they have no consequence on the expression of genes or proteins

51
Q

why is the intervening sequence region hypervariable?

A

-there is no selective pressure for genetic changes in this region because they do not impact the expression of genes or proteins

52
Q

what is a mutation?

A

genetic variations that actually cause disease

53
Q

what are the two things that one or the other needs to happen in order to indicate a RFLP?

A

-the genetic change needs to create or destroy a restriction site
ORRRRR
-the genetic change needs to have more or less of a repeated sequence

54
Q

what does RFLP stand for?

A

Restriction Fragment Length Polymorphism

55
Q

what accounts for 90% of genetic variation?

A

single nucleotide polymorphisms (SNPs)

56
Q

when would a single nucleotide polymorphisms (SNPs) cause a RFLP and when is it just a SNP?

A
  • when it creates or abolishes a restriction site, it will create a RFLP
  • when it doesnt, it is just a genetic varation
57
Q

is an RFLP from a SNP usually created by a disease causing mutation?

A
  • it can be

- more often its a harmless change that results in a different restriction pattern

58
Q

with SNPs, is the new fragment longer or shorter?

A
  • it depends!!
  • it can create OR abolish a restriction site
  • also depends on the original size of the fragment
59
Q

how are RFLPs created with SNPs?

A

-creating or abolishing a restriction site

60
Q

how are RFLPs created with VNTRs?

A

-more or less of a tandem repeat

61
Q

what is the size of the RFLP dependent on when created by a SNP?

A

-depends on different restriction cutting

62
Q

what is the size of the RFLP dependent on when created by VNTRs?

A

-depends on the number of repeats

63
Q

what are RFLPs created by SNPs used for?

A
  • used to mark genes

- disease markers

64
Q

what are RFLPs created by VNTRs used for?

A
  • used as molecular fingerprints

- used in forensics and paternity tests to identify individuals

65
Q

how does DNA cloning amplify DNA?

A

-amplify fragments by inserting them into a vector and the HOST REPLICATES the vector

66
Q

how does PCR amplify DNA?

A
  • amplification all done completely in a tube

- amplify DNA without having to clone it

67
Q

what do we need to create a primer for PCR?

A

-we need to know the sequence of two small flanking regions of the sequence we want to amplify

68
Q

what are primers (used in PCR)

A

-nucleotides that are complementary to the flanking region of the target DNA

69
Q

what enzyme do the primers for PCR act as a primer for?

A

DNA polymerase

70
Q

what is used to denature the DNA for PCR?

A

Heat

71
Q

what are the three steps of PCR that are repeated until the chain is the length you want?

A
  • denature
  • anneal
  • extend
72
Q

what are the general temperatures for denaturing, annealing, and extending?

A
  • denaturing: 95 C
  • annealing: 55 C
  • Extension: 72 C
73
Q

what kind of enzymes are used for PCR

A

heat stable Taq DNA polymerases

74
Q

what are the two major advantages of PCR?

A
  • high sensitivity: only need trace amounts of DNA to do it
  • speed: only takes a few hours
  • PCR gives really clean DNA
75
Q

what are the two methods used for analysis of gene expression?

A
  • northern blot

- microarrays

76
Q

what is a microarray and what is used for?

A
  • contain thousands of immobilized sequences (probes) on glass slides
  • used to compare global gene expression changes in different cells types
77
Q

what are the three ways to analyze proteins?

A
  • proteomics
  • enzyme-linked immunosorbent assays
  • western blot
78
Q

what is a major fault in ELISAs?

A
  • they can produce false positives
  • if negative, you can be confident in your results
  • if positive, you should re check with western blotting
79
Q

between western blotting and ELISAs, which one is most sensitive and which one is more specific?

A
  • ELISA is more sensitive

- western blotting is more specific

80
Q

what techniques are quantitative as well as qualitative?

A
  • Northern blotting
  • Western blotting
  • Microarray
  • ELISA
  • Protenomics
81
Q

Sickle cell is a mutation in what gene, and does what?

A
  • mutation in B-globin gene

- eliminates a restriction site

82
Q

sickle cell is an exception, but usually an RFLP used for diagnosis is linked to the disease, but not __________-

A

-no the actual disease causing mutation

83
Q

what are the three ways to test for sickle cell?

A
  • southern blotting
  • PCR
  • ASO probes
84
Q

what are ASO probes?

A
  • allele specific oligonucleotide
  • they recognize allele specific differences
  • hella specific
85
Q

why is ASO awesome?

A
  • specific and reliable
  • very fast
  • non-invasive
  • inexpensive

Biochem 4 (4 decks)

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