III. Short questions

Q1. How nucleic acids can be estimated by UV spectroscopy?

Ans. The amount of nucleic acids in a sample can be estimated by its absorbance at a wavelength of 260nm (in the UV region). Purines and pyrimidines have absorbance maxima slightly below and above 260 respectively. Thus the absorbance maxima of different fragments of DNA or RNA vary depending on their nucleic acid composition. Contaminants like proteins exhibit two absorbance peaks, one between 215-230 nm (due to peptide bonds absorption) and at about 280 nm (absorption by aromatic amino acids-tyrosine, tryptophan and phenylalanine).

Sample	Absorbance value	Quantity ( approximate )
Double-stranded DNA	1at 260 nm	50 μg/mL
Pure single-stranded DNA	1 at 260 nm	33 μg/mL
Pure RNA	1 at 260 nm	40 μg/mL
Pure protein (vary in general)	1 at 280 nm	1 mg/mL

 

Q2. What are the basic steps involved in polymerase chain reaction (PCR)?

Ans. The steps involved in basic polymerase chain reaction (PCR) are as follows :

a) Initial Denaturation- Denaturation of the template double stranded DNA to single strands at a temperature of 94–96°C for 7-10 minutes.

b) Denaturation- Heating the reaction mixture to 94–98°C for 20–30 seconds. It results in melting of the double stranded DNA by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

c) Annealing- Following denaturation, the primers are allowed to anneal to the single-stranded DNA templates at 50–65°C for 40–60 seconds. Typically the annealing temperature should be about 3-5 °C below the T m of the primers.

d) Extension- Extension/elongation step includes addition of dNTPs to the 3' end of primer with the help of DNA polymerase enzyme at a certain temp of 68-72°C for 50sec-1minute.

The above three steps (Denaturation, annealing and extension) are repeated for 30-35 cycles.

e) Final extension- This step is performed for 5–15 minutes at a temperature of 70–74°C after the last PCR cycle to ensure amplification of any remaining single-stranded DNA.

Final hold step at 4°C may be done for short-term storage of the reaction mixture.

Q3. How the DNA can be estimated in quantitative PCR?

Ans. Generally, two detection chemistries are used in qPCR:

a) Use of intercalating dye like SYBR® Green I dye which incorporates between the base pairs of DNA. This detection method is suitable when the PCR reaction generates a specific product, as the dye is capable of intercalating into any double-stranded DNA product. In the bound state, SYBR Green I exhibits 1000 fold more fluorescence than the unbound state. The fluorescence signal increases in proportion with the increase in amplified DNA.

b) Other detection chemistry includes TaqMan ^® probe, Molecular Beacons or Scorpion primers. In case of molecular beacons, they are labeled with a fluorescent dye or quencher and do not exhibit any significant fluorescence in the free, unhybridized condition. But upon binding to the template, the probe becomes fluorescent as the quencher gets distanced from the fluorescent reporter. The amount of PCR product amplified is directly proportional to the amount of fluorescence.