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Cell and Molec > SLIDES: Methods > Flashcards

SLIDES: Methods Flashcards

1
Q

What is the function of dATP, dGTP, dCTP, and dTTP in PCR?

A

They’re all deoxyribonucleoside triphosphates. It’s just a ton of the four ingredients for making nucleotides

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2
Q

Why do you have an mRNA/cDNA intermediate when you’re making cDNA?

A

Reverse transcriptase makes a compliment to the original mRNA strand. The intermediate is there because you haven’t built the compliment yet to that compliment yet

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3
Q

Where do HIV particles live in blood? Why is this relevant to diagnostic PCR?

A

In blood plasma.

Relevant because you centrifuge > then get RNA from just plasma

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4
Q

You’re at the last step of diagnostic PCR and put your sample on a gel. What else do you need on the gel?

A

Control using blood from noninfected person

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5
Q

What is the function of the dNTPs (dATP, dGTP, dCTP, and dTTP) in PCR?

A

They’re all deoxyribonucleoside triphosphates. It’s just a ton of the four ingredients for making nucleotides

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6
Q

You’re at the last step of diagnostic PCR and put your sample on a gel. What else do you need on the gel?

A

Control using blood from noninfected person

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7
Q

What does ddNTP stand for?

A

dideoxyribonucleoside triphosphate

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8
Q

If you want to sequence RNA, what do you generally do to it first?

A

Convert it to cDNA first > use methods for cDNA sequencing

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9
Q

How many reading frames are there for a piece of double-stranded DNA?

A

6 (3 for each strand)

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10
Q

How does manual sequencing work?

A

Make a bunch of gene copies in 4 test tubes > break by a specific letter (A,T,C,G) in each tube > put them all on the same gel to get a sequence

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11
Q

How does manual sequencing work?

A

Make a bunch of gene copies in 4 test tubes > break by a specific letter (A,T,C,G) in each tube > put them all on the same gel to get a sequence

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12
Q

How does automated sequencing work?

A

Add fluorescent-labeled ddNTPs to one tube > load on to gel > each nucleotide gets its own color > computer generates sequence with a camera

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13
Q

How does automated sequencing work?

A

Add fluorescent-labeled ddNTPs to one tube > load on to gel > each nucleotide gets its own color > computer generates sequence with a camera

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14
Q

If you have a random double-stranded DNA sequence, how do you know which of the 6 is the right reading frame?

A

The right one doesn’t have random stop codons in it (normal amount of stop codons = 1/20 codons)

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15
Q

If you have a random double-stranded DNA sequence, how do you know which of the 6 is the right reading frame?

A

The right one doesn’t have random stop codons in it (normal amount of stop codons = 1/20 codons)

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16
Q

What is recombinant DNA?

A

Somebody has manually added a sequence to it

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17
Q

What can you do to make a shitload of protein of interest? (uses expression vector)

A

Make recombinant DNA with a mega active promoter on it, makes a shitload of protein get made

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18
Q

How can you figure out amino acid sequence of a protein?

A

Use mass spec on it

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19
Q

How do you make the gene from an amino acid sequence?

A

BLAST sequence > synthesize primers > PCR

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20
Q

How do you make protein from cDNA sequence?

A

Put into expression vector with overactive promoter > put into host cell > make protein

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21
Q

How do you make protein from cDNA sequence?

A

Put into expression vector with overactive promoter > put into host cell > make protein

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22
Q

How do you go from protein > gene > protein?

A

Protein > mass spec > BLAST > DNA primer > PCR > GENE!! > expression vector > put in host > PROTEIN!!

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23
Q

How do you go from protein > gene > protein?

A

Protein > mass spec > BLAST > DNA primer > PCR > GENE!! > expression vector > put in host > PROTEIN!!

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24
Q

How do you make more of a protein?

A

expression vector w/ promoter, have host cell make protein

25
Q

how do you make more of a gene?

A

PCR

26
Q

how do you figure out amino acid sequences?

A

mass spec

27
Q

how do you replace one gene with another?

A

CRISPR

28
Q

What two things does guide RNA need in the CRISPR system?

A
  • Section to bind to Cas9

- Section designed by experimenter that binds to target DNA sequence

29
Q

What two things does guide RNA need in the CRISPR system?

A
  • Section to bind to Cas9

- Section designed by experimenter that binds to target DNA sequence

30
Q

What is PAM in CRISPR?

A

Sequence on 5’-3’ strand to distinguish normal genome and virus genome

31
Q

How do you edit multiple genes simultaneously?

A

CRISPR - just put CAS-9 in with multiple guide RNA sequences

32
Q

How do you edit multiple genes simultaneously?

A

CRISPR - just put CAS-9 in with multiple guide RNA sequences

33
Q

How many genes can CAS-9 fused to an activator turn on?

A

All of them

34
Q

How many genes can CAS9 fused to a repressor turn off?

A

All of them

35
Q

Why does RNAi work by injestion?

A

All you need is miRNAs or snRNAs, once those are in the body they knockout any mRNA you want

36
Q

What does RNAi introduce to a cell?

A

Double-stranded RNA that gets diced and binds to RISC

37
Q

Can you inactivate target genes with RNAi immediately after introduction?

A

No, you have to wait for it to spread through the organism

38
Q

How can you tell which cis-regulatory sequences make a gene get expressed in which cells?

A

Switch out a normal gene with a reporter gene, then make test DNA which doesn’t have some of the regulatory sequences. Look at where expression happens.

39
Q

What is an example of a reporter phenotype?

A

GFP

40
Q

How can you figure out when and where a gene is expressed?

A

in situ hybridization

41
Q

What does ‘hybridization’ in in ‘in situ hybridization’ refer to?

A

Two DNA molecules pairing back together when temperature lowers from 90C

42
Q

What is a DNA probe?

A

Short single-stranded DNA complimentary to a sequence of interest (‘target sequence’)

43
Q

What does ‘in situ’ in ‘in situ hybridization’ refer to??

A

‘in place’.

44
Q

How can in situ hybridization determine if RNA is there?

A

Put complimentary sequences into a cell. When they bind, you know the RNA is being expressed.

45
Q

What is qRT-PCR useful for?

A

Figuring out how many RNA samples you have

46
Q

How does qRT-PCR work?

A

It’s just normal PCR but the RNA has a fluorescent tag. Watch the amount of light it gives off grow in real time

47
Q

How could you tell on a graph which of two mRNA sequences you had more of with qRT-PCR?

A

The one you had more of will fluoresce earlier (starts to fluoresce at X sequences, if you have more sequences in the beginning you hit X faster)

48
Q

What are microarrays made of?

A

Glass microscope slides with hundreds of thousands of DNA fragments

49
Q

How are microarrays useful?

A

You can figure out which genes in the entire genome are being expressed at a given time

50
Q

What’s the obvious drawback to microarrays?

A

You need to know the sequences of mRNA samples in advance

51
Q

What do you do to make cDNA from mRNA?

A

1) Lyse and purify mRNA
2) Prime poly-A tail with poly-T prime3
3) Make cDNA with reverse transcriptase
4) degrade RNA with RNAse
5) Use DNA polymerase to synthesize second strand (remaining RNA fragment as primer)

52
Q

What fluoresces in microarrays?

A

cDNAs you attach

53
Q

a gene is highly conserved between mice and birds, what technique is most appropriate to compare the amount of expression of the gene in the livers of these animals?

A

In situ hybridization? or maybe qRT PCR?

54
Q

What does ChIP stand for?

A

Chromatin Immunoprecipitation

55
Q

question does ChIP answer?

A

‘What regulatory sequence does this protein bind to?’

or

‘what transcription factor/regulator is responsible for this pattern of gene expression’?

56
Q

Why do you need to break up DNA in ChIP?

A

To be able to pull specific parts bound to antibodies out

57
Q

Why does ChIP need formaldehyde?

A

To link proteins with regulatory sequences; sometimes the sequences are turned off and you want them all bound

58
Q

What all the techniques we learned and how are they useful?

A

{PCR : make more sequences}

{Manual/automatic sequencing : figure out what’s in your sequences}

{Expression vector (recombinant DNA techniques) : make more proteins}

{Mass Spec : figure out what’s in your amino acid sequences}

{BLAST : figure out what gene your amino acid sequences is built from}

{CRISPR : Replace one DNA sequence with another, turn whatever gene you want on/off }

{RNAi : Destroy RNA}

{Reporter genes : Figure out what regulators affect which genes where}

{in situ hybridization : see where sequences are in the cell}

{qRT-PCR : See how many RNA sequences you have}

{microarrays : See what sequences in a genome are active}

{ChIP : Figure out where in genome regulators are}

Cell and Molec (21 decks)

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