Question

(20 pts total) You are determining the Tm (melting temperature) for the double stranded DNA molecule created by hybridization of 2 oligonucleotides with the following sequences: O1 : 5’-d-GCTTGATTAGTTATTGC-3’ O2 : 5’-d-GGGGCAATAACTAATCAAGC-3’

a. (5 pts) Write down the sequence of the hybridized/annealed double stranded DNA molecule. Make sure the sequences are aligned, as they would be in the resulting DNA molecule. Label the 5’ and 3’ end of each strand. O1/O2

5'-d-GCTTGATTAGTTATTGC-3'

3'-CGAACTAATCAATAACGGGG-d-5'

b. (7 pts) You want to label this short piece of DNA with a radioactive isotope of 32 phosphorous, P . To do so, you can use a polymerase to fill in the overhang in the hybridized DNA in a. above. Draw the nucleotide tri-phosphate you would use for this purpose, clearly indicating which phosphate should be labelled with the P32 isotope and write down the sequence of the resulting double stranded DNA, as instructed for a. above. Labeled nucleotide: O1*/O2

c. (5 pts) You perform an experiment to generate a melting curve for the two DNA molecules in a and b above. Draw the two melting curves you would expect for the DNA molecules in the box on the next page, overlaid on the same graph, clearly labeling the dependent (Y) and independent (X) variables in the graph, the melting temperature, and which would be O1/O2 and O1*/O2. Explain your rationale briefly.

I NEED PART B AND C

Answer 1

(B) Radiolabeled nucleotides are commonly used for detection of specific nucleic acid sequences. They are typically incorporated enzymatically into DNA and RNA sequences for detection and analysis. Labeled nucleotides may be incorporated by a variety of methods including in vitro transcription with SP6, T3 or T7 RNA polymerase, 3’ end labeling with terminal deoxynucleotidyl transferase (TdT), T4 DNA polymerase or T7 DNA polymerase, random primed DNA labeling with Klenow fragment, cDNA labeling with AMV or M-MuLV reverse transcriptase, nick translation labeling with DNAse 1 and DNA Polymerase 1, and PCR labeling with thermophilic DNA polymerases like Taq or Pfu. The resulting labeled probes may then be used in applications such as in situ hybridization, microarray analysis, DNA sequencing, southern blotting and northern blotting.

DNA and RNA labeling teqniqe :-

Oligonucleotides can be labeled at either the 3' or the 5' end. Using polynucleotide kinase and ATP-gamma-32P, the 5' end is labeled. Using terminal transferase and deoxynucleotide triphosphate labeled on the alpha phosphate, the 3' end is labeled. Traditionally, the isotope of choice has been 32P, however 35S and 33P have been used successfully. Use of 35S and 33P is especially useful when high resolution (as in in situ hybridization) or when long probe stability is needed.