Question

2. PCR amplification of the TAS2R38 gene a. The number of copies of the 303 bp sequence grows exponentially (1-2-4-8-etc) after each cycle. The number of cycles we used is on page 97. What is the number of copies of the 303 bp fragment that will theoretically be present at the end of our reaction? b. Denaturation of the 303 bp segment of the TAS2R38 gene is a critical first step in the PCR perties of a DNA segment that could alter the initial denaturation process. Name two pro temperature. c The optimal annealing temperature (Ta) is the range of temperatures where efficiency of PCR amplification is maximal and depends directly on length and composition of the primer(s). An annealing temperature 5°C below the lowest melting temperature (T) of the primer pair used 1s typically used. whatís the T "ofour primers? Tisdef ned as the temperature where 50% of double-stranded DNA is made single-stranded and is calculated for primers using the formula: Z T, 64.9°C + 41°C [(number of Gs and C's in the primer-16.4)/N] (N is length of t primer). 4. (Week 3) Gel electrophoresis of the PCR products to determine your genotype OUR APPROACH (IN MORE DETAIL) You will be using your own genomic DNA as a template to specifically amplify a 303 bp region of the TAS2R38 gene using provided primers. This region contains SNP785 so you can determine your genotype with respect to tasting PTC. You will set up an ampli- fication reaction of your TAS2R38 gene and place it in the thermocycler, an instrument that automatically adjusts temperatures and incubation times needed for amplification of the 303 bp segment of your PTC (TAS2R38) gene. The reaction settings of the thermocycler are as follows: 1st Phase 2nd Phase 3rd Phase 1 cycle of 40 cycles of 1 cycle of 95 C for 10 minutes 55°C for 5 minutes 72°C for 90 seconds 95°C for 45 seconds 55°C for 45 seconds 72°C for 10 minutes 4°C untih collected A portion of your amplified DNA will then be subjected to the nu4H1 restriction endonuclease that cleaves DNA at the recognition site: Fnu4H1 5--GCNGC---3 3' CGN C G-"-5.

Answer 1

a)

During polymerase chain reaction (PCR), the number of double stranded DNA pieces is doubled in each cycle.

Therefore, after n cycles, total number of the copies of double stranded DNA would be 2ⁿ

So after 40 cycles, total number of 303 bp long template would be 2⁴⁰ copies= 1,099,511,627,776 copies

b)

To facilitate the separation of double stranded DNA into single stranded DNA, the initial denaturation step is carried out at the beginning of PCR. Initial denaturation steps aims to complete denaturation of the template DNA.

The initial denaturation step is commonly performed at 94–98°C for 1–5 minutes.

The temperature and time used for initial denaturation steps in PCR is greatly influence by the following two properties of template DNA

1) Nature of the template DNA: whether the template is a mammalian genomic DNA or a plasmid or a PCR product i.e. based on the nature of the template, the temperature for initial denaturation may vary. For example, as the mammalian genomic DNA is more complex and larger in size, it require longer incubation period than plasmid for initial denaturation.

2) GC content of the template DNA:

Template DNA with high GC content require higher temperature and longer incubation for initial denaturation compared to the DNA template having less GC content.

c) Suggestion

Generally the length of the PCR primers are 17- 30 bases. The bases in PCR primers may be counted i.e the number of A, T, G and C may be counted and as per the formula given in the question the Tm of the PCR primer (Detail of PCR primer is not available in the question) would be calculated.