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MDSC 408 > Quiz 1 - Lecture Component > Flashcards

Quiz 1 - Lecture Component Flashcards

1
Q

How do Fluorescent proteins get colour

A

The chromophore (molecule that absorbs light and he reflects colour) inside the barrel structure

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2
Q

Different chromophore =

A

different colour

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3
Q

What gives diff colours in a barrel

A

the amino acids in it

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4
Q

Chromophores are chemically altered without enzymes so using

A

oxidation or light or stuff

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5
Q

PCR is a rapid procedure for

A

in vitro enzymatic amplification of a segment of DNA

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6
Q

applications of PCR include 6

A

-cloning DNA or cDNA

-in vitro mutagenis / engeenering

  • genetic fingerprints of forensic samples

-assays fo infectious agents

  • Quantifying rna

-covid testing

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7
Q

Covid pcr testing ha real time

A

reverse trancription PCR of nasal swabs

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8
Q

RNA extracted would be

A

revers transcribes to cDNA

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9
Q

DNA pol wil adda nucleotide to the

A

3’ OH of the primer, therfore pol needs a primer with 3’oh

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10
Q

You need a primer for

A

each strand

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11
Q

The primers need to be tightly bound to

A

the template at 3’ end but u can have some slack on the 5’ end

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12
Q

in the case that dna is denatures, in a new repli the forward primer anneals to

A

the lower strand and the reverse primer anneals to the upper strand.

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13
Q

PCR Steps 6

A
  1. Initial melting at 94 C for ~5 min
  2. Denaturation of the DNA strands (94-95 C)
  3. Annealing of oligonucleotide primers (55-65 C)
  4. Extension/polymerization with polymerase (72 C)
  5. Repeat steps 2-4 for 25-30 cycles
  6. Final extension at 72 C for 5-10 min
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14
Q

Desires amplified products appear during the 3rd cycle and the product accumulate

A

exponentially

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15
Q

equation?

A
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16
Q

Taq is a

A

thermostable dna-dependent dna pol and thought to be stable cuz of hydrophobicity at the core of the enzyme and stabilization of electrostatic forces

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17
Q

Taq has 5’ -> 3’’ pol activy but also

A

5’-> 3’ exonuclease activty but ni 3’ -5’ exo so no proofreading

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18
Q

taqs substrate is

A

ssdna, primer with 3’oh

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19
Q

fidelity if the

A

error rate per nucleotide

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20
Q

3’-5’ exo provides

A

proofreading by removing 3’ terminal mismatches

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21
Q

Primers determine the specificity of amplification and are designes to

A

minimize amplification of unwanted sequences

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22
Q

Primers in length are 18-24 nt and pairs do not differ by over

A

3 nt

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23
Q

Primer dont gave a

A

secondary structure (so like hairpin loops)

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24
Q

No primer

A

primer interaction at 3’ end???

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25
Q

Magnesium ions are important too

A

cuz al thermostable pols require free divalent cations for activity, usually bg++ (dntps and primer bing mg++)

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26
Q

Nucleotides like datp, dctp, dgtp and dttp sre important and in high [ ]

A

are limitjng probs cu of sequestering mg ++

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27
Q

The template dna can be

A

ss or ds, liner or circular

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28
Q

Denaturations is determined by

A

tm cuz if temp not high enough AT will denature but CG wont

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29
Q

In annealing if tempo too high no annealing and if too low

A

non-specific amplification can occur, usually tm05 used to ensure specificty

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30
Q

What is tm

A

The melting temperature (Tm) is the temperature at which 50% of the double-stranded DNA is changed to single-stranded DNA.

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31
Q

Extension is at or near

A

optimum temp for pol

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32
Q

The number of cyckes depns on

A

initial copy number, efficieny of amplification

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33
Q

Why do we need primers for PCR

A

Cuz they need a hydroxy end so the 3’ end

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34
Q

SOE is

A

splicing by overlapping

  • Site specific mutagenesis
  • Creation of chimeric molecules
  • Assembly of large segments of DNA
  • For making deletions
  • For introducing insertions
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35
Q

Explain PCR

A

you need primers ABCD, b and c put the actual mutation for both directions and AD are for growing out both ends (flank), they can aslo be used for recognition sites for enzymes,

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36
Q

Primers always become

A

part of the product of PCR

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37
Q

The worst place to put a primer is

A

the 3’ end but the middle of 5’ end is fine

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38
Q

If you add all the primers together you’ll just get

A

all the AD together and bc togethr cuz theyre complimentary and it wont work

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39
Q

I dotn get it continue later

A

review slides

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40
Q

so the melting temp of primers is determined by

A

their size and GC content

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41
Q

lets say we have a primer with a high tm but the revers primes tm is low, what can we do?

A

Design a longer primer, cuz the longer the higher the temp

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42
Q

BC primers are how long?

A

18-35 nucelotides

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43
Q

They also provided adequate

A

overlap for efficient splicing

44
Q

Flanking primers, AD are also just so imprtant cuz they can

A

be used to introduce recognition sites for enzymes

45
Q

Its not good to have enzyme cut sites

A

too close to the ends , why?

46
Q

The length og these primers is

A

18-35 but at least 10 nts

47
Q

Deletions work by creating

A

hairpin loops, so its not transcribed and therefore then not replicatd when it goes up exponentially

48
Q

The forward primer will begin

A

at the 5’ end of DNA a

49
Q

Recombinant DNA is

A

any DNA molecule made up of sequences/fragements from diff sources

50
Q

DNA cloning is

A

preparing large numbers of identical dna molecules

51
Q

DNA clong is made possible by

A

using recomb dna methods

52
Q

You can clone DNA to study genes , structurs, functions and how they are regulated, to do this

A

you need to isolate the gene and put it into a vehicle or vector to make more recomb DNA? then tranfer thsi to a hos vell which eill replicate with the recomb DNA, WITH THIS IT AMPLIFIES

53
Q

bACTERIAL dna IS

A

methylated at certain site and these are charctersitic of th species

53
Q

This addition of methyl sites

A

protected the sites from sesive attacks by restricition enzymes

53
Q

SLIDE 16, 17, 18

A
54
Q

Methylation patterns are

A

maintianed during dna repli

55
Q

foreign dna that is unmethylated or has diff methylation pattern than host cell

A

will be degraded

56
Q

Types I and III are

A

not very usefull cuz they cleave DNA at sites away from the recognition site and have random cleavage patterns

57
Q

Type II however

A

recognizes specific sites and cleaves those sites

58
Q

Nomeclature

A

1st letter of genus name, 1st 2 letters of species name, strain types, nth enzymes of that types

59
Q

Type II restriciton enzymes recoignize

A

symmetric DNA sequences 4-8 bp

60
Q

Theyre usually pallindromic

A

so weather it reads 5’ to 3’ or 3’ to 5’ its the same

61
Q

They can create either

A

either sticky ends or blunt ends

62
Q

Hindiii makes

A

sticky ends

63
Q

Smal makes

A

blunt ends

64
Q

Ecoti makes

A

sticky ends

65
Q

BamHI makes

A

sticky ends

66
Q

Practice slide 25

A
67
Q

Ligase catalyzes the

A

formation of a phosphodiester bond between the 5’ and 3’ group

68
Q

What does it require as a cofactor

A

ATP

69
Q

Why is dna ligase needed

A

cuz when restriction enzymes cut they maker hydrogen bonds to make bade paiors but arent covalently bonded ?

70
Q

Chimeric restrictions sites

A

?

71
Q

DNA vectors can

A

replicate autonomously in bacteria

72
Q

They contain unique…

A

restriction endo nuclease cleavage sites that are present only once in the vector

73
Q

What do they have to distinguish host cell that has the vector vs those that do

A

a selectable marker like antibiotic resistance genes

74
Q

They have to be easy to seperate from the

A

bacterial chromosomal DNA

75
Q

Compare the Basis, size limits and Use of Plasmids

A

naturally occuring;
Under 10 KB;
Subcloning and downstream manipulation, cDNA cloning and expression assays;

76
Q

Compare the Basis, size limits and Use of Phages

A

Bactiriophage lambda

5-20kb

Genomic DNA cloning, cDNA cloning and expression libraries

77
Q

Compare the Basis, size limits and Use of cosmid

A

Plasmid containing bacteriophage lambda cos site

35-45kb

genomic library contruction

78
Q

Compare the Basis, size limits and Use of BAC

A

Escherichia coli f FACTOR PLASMID

75-300 KB

Analysis of large genomes

79
Q

Explain plasmids

A

their naturally occurring extra chromosomal double- stranded circular DNA that carry an origin of repli

80
Q

One of the first plasmids developped for cloning was

A

pBR322

81
Q

It was modified to have

A

an antibiotic resistance gene and multiple cloning sites so basically you can linearize and add insert

82
Q

What is a very important thing u need to put these inserts in a vector

A

U need a ligase to seal it shut

83
Q

Then you transfer the ligation reaction to a

A

bacterial host and thats where it multiplies

84
Q

Why use antibiotics

A

Cus the successful ones with the plasmid will have the antibiotic gene and survive while the ones that didn’t take up the plasmid with die

85
Q

How does ligation work in this

A

forms phosphodiester bonds between the 5’ phosphate and the 3’ hydroxy

86
Q

So why isn’t ligation 100% effective?

A

cuz the to ends can easily go back together

87
Q

How can we fix the problem of ends sticking back together without the vector?

A

Using a phosphatase so removing 5’ phosphate so that is can join back together and have the insert DNA provide a 5’ phosphate.

88
Q

A second way to avoid ends going back together is to

A

Cut with 2 different restriction enzymes to make non complimentary sticky ends

89
Q

So now, u only have the option of having the same restriction enzyme cut, what the issue here

A

that it could be put in either direction (cuz palindromic?)

90
Q

How do we make sure thats not happening

A

with gel electrophoresis, so basically if it cuts in the write spots you’ll get certain bands and if it cuts in the wrong direction you’ll get different one (cuz you put a certainrestriction enzyme within you insert toom tocheck maybe?)

91
Q

So only transformed bacteria will survive but how do you know which one has recombinant DNA (the insert)?

A

blue-whote screening so ones with the vector will be white while others will be blue (without)

92
Q

So technically this works cuz of lac z , explain

A

so the e-coli has the n-terminal of lac z, and the plasmid will have the N-terminal so when they are together they work and have functional b-galactosidase enzyme, if there’s cleavage of x-gal - blue so it has plasmid, if there is white it doesn’t have plasmid cuz it didn’t cleave anything

93
Q

restriction enzyme have specific bufferes and they differ by

A

salt, mg, pH and buffer type

94
Q

If doing directional cloning it is best to

A

have enzymes that work together in the same buffer

95
Q

How does the buffer chart work

A

u want to use a buffer that’ll work on both so nearly 100%, if you cant find one like that you’d have to do it sequentially so using one enzymes buffer and then the others

96
Q

How can u use PCR to add in restriction sites

A

slide 8

97
Q

Taq has terminal transferase activity so

A

that adds a single adenosine to the 3’ end of the ThPCR amplified fragment

98
Q

The 2 types of topoisomerase are

A

types 1 which cuts 1 strand and type 2 which cuts both strands

99
Q

TA cloning uses

A

types 1 topo

100
Q

The 3 main components of ta cloning are

A

3’ a overhang on both ends of the taq amplified PCR
vector with 3’ T overhangs
t4 DNA ligase

101
Q

However, pols that have 3’ - 5’ exonuclease activity or proofreading wont do this so

A

these products would have to be blunt ended ligated into vectors or As added with taq pol after pcr

102
Q

Topo q bind to duplex DNA at specific sites and

A

cleaves the phosphodiester backbone after 5’CCCTT in a strand

103
Q

The energy from the broken bond is conserved and does what

A

its conserved by the fromartion of a covalent bond between 3’ phosphate and the cleaved steandf and tyr-274 on topo 1.

104
Q

The phospho-tyrosyl bnd can be attacked by the 5’ oh

A

of the og cleaved stran to reverse the reaction and release the topo 1 enzyme

105
Q

What does TA cliing not require

A

a ligase

MDSC 408 (2 decks)

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