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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
how do you make more of a gene?
PCR
26
how do you figure out amino acid sequences?
mass spec
27
how do you replace one gene with another?
CRISPR
28
What two things does guide RNA need in the CRISPR system?
- Section to bind to Cas9 | - Section designed by experimenter that binds to target DNA sequence
29
What two things does guide RNA need in the CRISPR system?
- Section to bind to Cas9 | - Section designed by experimenter that binds to target DNA sequence
30
What is PAM in CRISPR?
Sequence on 5'-3' strand to distinguish normal genome and virus genome
31
How do you edit multiple genes simultaneously?
CRISPR - just put CAS-9 in with multiple guide RNA sequences
32
How do you edit multiple genes simultaneously?
CRISPR - just put CAS-9 in with multiple guide RNA sequences
33
How many genes can CAS-9 fused to an activator turn on?
All of them
34
How many genes can CAS9 fused to a repressor turn off?
All of them
35
Why does RNAi work by injestion?
All you need is miRNAs or snRNAs, once those are in the body they knockout any mRNA you want
36
What does RNAi introduce to a cell?
Double-stranded RNA that gets diced and binds to RISC
37
Can you inactivate target genes with RNAi immediately after introduction?
No, you have to wait for it to spread through the organism
38
How can you tell which cis-regulatory sequences make a gene get expressed in which cells?
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
What is an example of a reporter phenotype?
GFP
40
How can you figure out when and where a gene is expressed?
in situ hybridization
41
What does 'hybridization' in in 'in situ hybridization' refer to?
Two DNA molecules pairing back together when temperature lowers from 90C
42
What is a DNA probe?
Short single-stranded DNA complimentary to a sequence of interest ('target sequence')
43
What does 'in situ' in 'in situ hybridization' refer to??
'in place'.
44
How can in situ hybridization determine if RNA is there?
Put complimentary sequences into a cell. When they bind, you know the RNA is being expressed.
45
What is qRT-PCR useful for?
Figuring out how many RNA samples you have
46
How does qRT-PCR work?
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
How could you tell on a graph which of two mRNA sequences you had more of with qRT-PCR?
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
What are microarrays made of?
Glass microscope slides with hundreds of thousands of DNA fragments
49
How are microarrays useful?
You can figure out which genes in the entire genome are being expressed at a given time
50
What's the obvious drawback to microarrays?
You need to know the sequences of mRNA samples in advance
51
What do you do to make cDNA from mRNA?
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
What fluoresces in microarrays?
cDNAs you attach
53
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?
In situ hybridization? or maybe qRT PCR?
54
What does ChIP stand for?
Chromatin Immunoprecipitation
55
question does ChIP answer?
'What regulatory sequence does this protein bind to?' or 'what transcription factor/regulator is responsible for this pattern of gene expression'?
56
Why do you need to break up DNA in ChIP?
To be able to pull specific parts bound to antibodies out
57
Why does ChIP need formaldehyde?
To link proteins with regulatory sequences; sometimes the sequences are turned off and you want them all bound
58
What all the techniques we learned and how are they useful?
{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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