Lecture 8: Nucleic Acids

Question 1

What is metagenomics?

Answer 1

The study of the structure and function of entire nucleotide sequences isolated and analyzed from all the organisms. Can be used to…
1. Find reservoir host species for newly discovered viruses
2. Track evolution and geo-phylogeny in real time

Question 2

Define nucleic acid sequencing

Answer 2

Using RNA and DNA to sequence genomes.

Question 3

Define molecular virology and its relevance

Answer 3

It involves proteins and nucleic acids. Molecular virology comprises a large portion of virology.

Question 4

How is a PCR reaction executed?

Answer 4

Polymerase Chain Reaction. Exponential amplification of a known DNA segment is done by adding two defined DNA primers, a forward and reverse primer. The template DNA is denatured, annealed with primers, and elongated in one complete cycle. (n) number of cycles will give 2^(n) copies of the template DNA.

Question 5

How is RT-PCR done?

Answer 5

Reverse Transcription - Polymerase Chain Reaction. A single mRNA strand is used as a template to synthesize the first strand of DNA using reverse transcriptase. From here, PCR steps.

Question 6

How can PCR be used in virology diagnostics? What are its limitations?

Answer 6

Used to prove the presence or absence of some DNA. Primers can match to a known segment of viral DNA (ie. HSV). Then, use these primers in a PCR reaction in cell biopsies (ie. mouse ganglion). Check for PCR products of predicted size by gel electrophoresis through agarose gel. This method cannot quantify amount of product.

Question 7

How does real-time-PCR work to quantify nucleic acid product? What is the threshold, endpoint, and exponential range?

Answer 7

PCR reaction, only difference is that only products are fluorescent. Exponential amplification will hit an endpoint signal after 44+ cycles after dNTPs are exhausted. The exponential range is the area of PCR amplification between the endpoint and threshold. The faster a PCR reaction reaches the threshold fluorescent signal strength, the more template DNA there is present in the original sample.

Question 8

How does the relative quantity of a PCR reaction differ if one reaction undergoes 22 cycles and another undergoes 23?

Answer 8

There’s half as much DNA product in the 22-cycle reaction than in the 23-cycle reaction. 2^(n) where (n) is the number of cycles.

Question 9

How is PCR cycles related to amount of starting template?

Answer 9

The number of cycles required to achieve some particular product signal strength less than the endpoint is really proportional to amount of starting template.

Question 10

What are PCR tests not able to do?

Answer 10

Differentiate btw infectious and non-infectious viruses

Question 11

PCR input material?

Answer 11

Nasal or throat swab

Question 12

Advantages of PCR tests?

Answer 12

Early availability

Question 13

Disadvantages of PCR tests?

Answer 13

Lab instruments required

Question 14

How can false positives of PCR tests happen?

Answer 14

PCR is highly sensitive. Multiple primer sets tested in parallel-highly specific. It’s possible that some junk got amplified.

Question 15

How can false negatives of PCR tests happen?

Answer 15

Because of extreme sensitivity, false negatives are unlikely

Question 16

How can PCR be used to create a mutant DNA strand?

Answer 16

Amplify overlapping regions with PCR primers with a mutated point. Need two sets of forward and reverse primers. This will create two separate segments amplified by PCR. Then, do a second PCR reaction with the tail primers of each segment to join them together. Product will have a mutagenized region.

Question 17

What is the dideoxy method of enzymatic DNA sequencing?

Answer 17

Do a PCR reaction with 5% dideoxynucleotides, ddNTPs, and 95% normal nucleotides, dNTPs. Then do four separate reactions, with each reaction having a different ddNTP (ddGTP, ddATP, ddCTP, ddTTP). Once ddNTPs are incorporated, PCR rxn halts. Use gel electrophoresis to see the length of each product from each ddNTP reaction, each lane corresponding to a nucleotide. Read from bottom up to get the entire sequence of template DNA.
In a different rxn, ddNTPs can be fluorescent to be read by a laser and will be read in real time by a laser.

Question 18

How can we use metagenomics in viral discovery?

Answer 18

Sequence a whole environment simultaneously. Start w/ environmental sample and either:

1. Isolate a community of viruses to filter, digest, and extract its nucleic acid, fragment it randomly, add adaptor to ends of genomes via litigation, then sequence in parallel.
2. Extract all nucleic acids in the environment, use PCR to pick out known products, then sift for gene mutations among the products.

Question 19

What are the three classes of viruses found in environments?

Answer 19

1. Known
2. Highly divergent
3. Unknowns (Most abundant)