Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Scientific Skills > Quantitation Of Nucleic Acids > Flashcards

Quantitation Of Nucleic Acids Flashcards

0
Q

What wavelength is DNA maximally absorbed ?

A

260nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
1
Q

What is the simplest method to quantify nucleic acids ?

A

Spectrophotometry

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

An absorbance of 1 at 260nm in a 1cm path length indicates what DNA concentration ?

A

50micrograms/ml

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is ten chemical constituent of DNA that contributes to the absorbance at 260nm ?

A

The bases because they are hexagonal and symmetrical so they have delocalised electrons around the ring which can absorb the UV light

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What molecules might contaminate a DNA sample ?

A

RNA because it also contains bases
Certain aromatic proteins such as tryptophan, tyrosine and phenylalanine - absorb light around 280nm but they also show significant absorbance at 260nm - the aromatic side chain at absorbes he uv light

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How are contaminants removed from a DNA sample ?

A

RNases remove RNA by degrading them but this causes th addition of protein
PolyT tails can be used to attach to the polyA tails of mRNA molecules to pull them out
To remove protein phenol and chloroform are added to remove the protein into a precipitate at the phenol water barrier
Or the DNA igself could be extracted using ion exchange binding resins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What effect does adding phenol to DNA sample have ?

A

It is an aromatic protein so it can absorb light at 270nm which would interfere with the assay so even though it is necessary for DNA purification it has to be applied with chloroform so it can be extracted

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How do you concentrate DNA in a dilute sample ?

A

Ethanol precipitation

Cooling it enhances the precipitation and then centrifuging it to get a pellet

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Absorbance methods are not actually that sensitive, what is another way of quantifying DNA?

A

Fluorescence methods
Adding a fluorescence dye such as sybr green because it is able to intercalate between the bases of double stranded DNA and then they can fluoresce

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What wavelength does protein absorb UV light ?

A

280nm absorbs it more strongly than DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is the A260:A280 ratio for pure DNA ?

A

2

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is the A260:A280 ratio for pure protein ?

A

0.57

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Why does the A260:A280 ratio decrease as protein concentration increases ?

A

Because proteins absorb light more at 280nm so if more protein is present the ratio will be closer to the pure protein ratio because more light will be being absorbed at 280nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are restriction endonuclease?

A

They are enzymes which break down DNA into smaller fragments
They recognise specific sequences in the DNA sequence at cut and their specific recognition site
Allowed the discovery of recombinat DNA technology and are useful for cloning genes and disease diagnosis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Which direction does DNA travel in ?

A

Travels towards the positively charged anode because DNA is negatively charged due to its phosphate group

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is translation and transcription?

A

Transcription - production of an mRNA strand from DNA

Translation- production of a protein at a ribosome from the mRNA strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are transcription factors ?

A

They control production of mRNA by binding to specific promoter regions

17
Q

What does controlling the abundance of mRNA allow ?

A

Regulate process such as development
Respond to stress
Adapt to altered circumstances

18
Q

What has to be extracted to measure the transcript abundance of a specific gene ?

A

Total RNA

19
Q

What is total RNA made up of ?

A

Ribosomal and transfer RNA -96%

mRNA - 4%

20
Q

Why is there more ribosomal RNA compared to mRNA ?

A

Because ribosomal population is much more fixed and static because we need lots of ribosomes to produce proteins
A single mRNA strand can be amplified by many ribs poems making a massive amplification of the signal

21
Q

Is RNA a stable molecule ?

A

No
Because it contains ribose sugar which has a hydroxyl group that acts as a nucleophile, increasing the hydrolysis of the molecule

22
Q

How is RNA degraded ?

A

By RNases

They are everywhere so upon extraction of RNA it must be protected to prevent its degradatio.

23
Q

What are protein denaturants and give examples

A

They destroy RNases

Phenol and guanidinium thiocyanate

24
Q

How is mRNA separated from rRNA?

A

mRNA has a polyA tail which means it has lots of As on the end of it
Therefore it can be hooked out with an oligonucleotide T
Oligonucleotide Ts bund to the As allowing the mRNA strand to be core over from the rRNA and tRNA

25
Q

What precautions should be taken when handling extracted RNA ?

A

Wear gloves to cover hands because they will have RNases on the,
All equipment must be decontaminated
Tissue could be frozen as enzymes don’t work as well at low temperatures
During processing of the sample it should remain on ice

26
Q

How can you measure the amount of a transcript fro a specific gene ?

A

Complementary sequences of nucleotide

27
Q

What techniques allow the interrogation of the expression of all gene at the same time ?

A

Microarrays

Throughput sequencing

28
Q

What is a microarray ?

A

An array of many thousands of spots each with a DNA probe for a single gene - DNA probe hybridises the product of a single mRNA exclusively

29
Q

Why are microarrays useful to scientists ?

A

They can compare the relative expression levels of genes in a healthy cell and a diseased cell or against a cell that’s had a drug administered to it

30
Q

Explain the microarray process for comparing genes in a cancer cell and a non cancer cell

A

Isolate RNA from both cells
RNA is reverse transcribed to produce cDNA probe in the presence of fluorescently labelled nucleotide (Cy3 and Cy5)
Fluorescently labeled cDNAs are mixed together so they hybridise on the microarray
Microarray can then be scanned and the fluorescence emissions at specific wavelengths can be quantified

31
Q

What is the levels of expression of a gene not an indication of ?

A

Not an indication of its biological importance

32
Q

What does the magnitude of changes to the expression of a gene not proportional to ?

A

The effects of those changes

33
Q

What does northern blotting measure ?

A

Measure mRNA transcript abundance

34
Q

What does southern blotting measure ?

A

Used to identify specific sequences of DNA

35
Q

Explain the process of northern blotting

A

Total RNA is run on agarose/polyacrylamide gel to separate RNA on basis of size
RNA fixed onto nitrocellulose membrane by cross linking
mRNA sequence of gene of interest will be sliced without its introns and compleat art probe produced
Probe is labelled with 32P-dCTP to cytokines with radioactive phosphorous
Probe is hybridised to the membrane and then exposed to film to produce black marks representing where the probe has bound to the mRNA species for the gene of interest

36
Q

In northern blotting what does the size and intensity of the marks indicate ?

A

Strong indication of relative quantities of mRNA

37
Q

What is normally used as a loading control in northern blotting ?

A

Actin because it tends to remain constant independent of changes to condition s

38
Q

What is e purpose of the control ?

A

To ensure the same amount of total RNA is loaded to each well
If there are changes in the mRNA transcript but actin stays the same then you know that the amount of mRNA for the gene of interest has changed
But if you see changes in the intensity of the actin band then you known the total RNA may not have been prepared properly

39
Q

What is reverse transcriptase PCR?

A

Process to amplify mRNA from small samples

Involves using a viral enzyme to produce a DNA transcript of mRNA

40
Q

Explain the cycle in PCR

A

Heating the double stranded RNA to separate it
Cooling it to allow the primers to bind
Allowing sufficient time to allow the DNA polymerase to extend the primer to produce a new full strand

Scientific Skills (8 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions