DNA in the Lab

Question 1

What is the absorbance of DNA and RNA? How is this used in spectrum experiments.

Answer 1

260nm. Likely the substances is a nucleic acid.

Question 2

What is the purpose of electrophoresis?

Answer 2

Provides data on different fluorescent peaks so you can work out what bases are in the double helix.

Question 3

What is a melt-curve?

Answer 3

Exploit DNA - only fluoresces when double stranded - can melt apart and check curve to endure get single PCR product.

Question 4

How do bases absorb UV light?

Answer 4

The conjugated ring structure, pi electrons absorb energy in the form of light and from this you can get an average of the four bases in a sequence of DNA.

Question 5

What has an absorbance of 1 for ssRNA and dsDNA?

Answer 5

50 micrograms/ml ds

40micrograms/ml ss

Question 6

What is the hyperchromic effect? Why does this occur?

Answer 6

SS nucleic acid molecule absorbs more UV light than DS. The ordered stacking of base pairs in ds DNA hinders photon access.

Question 7

What is the charge on the backbone?

Answer 7

Both strands of the backbone have a negative charge for each nucleotide.

Question 8

How can the negative charge on the backbone of nucleic acids be manipulated in the lab? What does migration mean?

Answer 8

In electrophoresis - subject them to an electric field and the nucleic acids will move to the positive electrode. The migration will depend on the number of bases in the DNA.

Question 9

What can gel electrophoresis be used for?

Answer 9

Separating DNA fragments by size. Larger molecules have more difficulty moving through the gel and so smaller, lighter fragments will move much faster toward the positive charge.

Question 10

What is Ethidium Bromide used for in the lab?

Answer 10

It intercalates with the dsDNA - slides between the bases. It fluoresces under UV light and so we can detect DNA by fluorescence.

Question 11

How does intercalation work?

Answer 11

The substance can fit into the double helix as it looks similar to a base pair and so can fit between the helix the way a base pair does.

Question 12

What techniques can be used to disrupt the double helix?

Answer 12

Pull it apart to make it ss (melting). Increase the temperature to disrupt the base stacking and increase the pH to remove the ring N protein from Thymine and Guanine, thus, preventing H bonding.

Question 13

What is denaturation of DNA? How can it be monitored?

Answer 13

The melting of dsDNA to ssDNA. It can be monitored through looking at absorbance at 260nm due to the hyperchromic effect -will see higher absorbance when ss (1.4 x higher).

Question 14

What is the melting temperature? Tm.

Answer 14

Midpoint of the transition from ds to ss. 50% ds 50% ss.

Question 15

What influences the Tm? Why?

Answer 15

The content of GC in the DNA sample. Higher content = higher melting temperature. GC has 3 hydrogen bonds - stronger and harder to melt.

Increasing the length.

Question 16

What is re-annealing. What is the temperature (Ta) for this to take place at?

Answer 16

SS to DS. Approximately 15 degrees lower than Tm.

Question 17

How is base pairing promoted?

Answer 17

Increasing ionic strength because the cations shield the phosphates and thus reduce the repulsive effect of the strong negative charges. Brings strands together more easily.

Question 18

What ion gives the best shielding capabilities for base pair promotion? Why don’t we use this ion?

Answer 18

Mg2+, don’t use it as they are necessary for reactions which hydrolyse the phosphodiester bond - use sodium.

Question 19

What does reducing temperature do for base pairing?

Answer 19

Lowers the entropy of reaction and facilitates the base pairing.

Question 20

What is PCR?

Answer 20

Polymerase chain reaction - allows us to amplify a specific section of DNA

Question 21

How is PCR performed?

Answer 21

Sections of DNA flanked by synthesised primers and are amplified many times (billions). Use a specific primer complementary to specified region of DNA to make copies of target sequence.

Question 22

What is added to a PCR tube?

Answer 22

Sample DNA, substrates (dNTPs), Primase (forward and reverse), taq polymerase and buffer.

Question 23

Why is taq polymerase used in PCR?

Answer 23

Has proteins that are not denatured at high temperatures so primers can access sequence once two strands are separated.

Question 24

Explain the process of PCR in depth.

Answer 24

Physical card 15

Question 25

What is the role of primers in PCR and how are they able to perform this role?

Answer 25

Primers flank (lie on either side of) the region of interest. They can do this because they are made uniquely to complement this region (need to be 18 nt long).

Question 26

What are the advantages and limitations of PCR?

Answer 26

Adv:
Quickly synthesise many copies of a target DNA sequence
Completely in-vitro - don’t need to grow anything
Can use low amounts of low quality DNA

Disadv:
Some information about target sequence must be known to pick the right primer
Sensitive to contamination

Question 27

What is dideoxy sequencing?

Answer 27

A form of DNA sequencing that exploits the fact that DNA polymerises require a 3’ OH for extension - this is where new nucleotides are added.

Question 28

How is dideoxy sequencing performed?

Answer 28

Card 16

Question 29

Why would we use reverse transcription in a lab?

Answer 29

For synthesising cDNA for qPCR, micro arrays and RNA sequencing.

Question 30

What is reverse transcription used for in cells?

Answer 30

Produced by retroviruses to make a complementary strand of DNA from their RNA genome upon infection

Question 31

Where do we start reverse transcribing from?

Answer 31

For mRNA - oglio dT - string of t’s base pair with poly A tail and will reverse transcribe the entire mRNA transcript.
For your gene of interest only - a specific primer.
Everything - random primer - nnnnnn- bind anywhere that 6n’s match 6 bases and reverse transcribes from that point.