Sequencing reactions
Sequencing reactions typically use only one type of primer. The primer
molecules anneal and provide a free 3′ OH group to which nucleotides can add. Nucleotides base pair with the template strand and are linked in a 5′ to 3′ direction by DNA polymerase. If double-stranded DNA is used as a template for DNA sequencing, the double-stranded DNA is denatured to yield single-stranded DNA.
a. How many identical molecules of DNA are present in a 0.5 ug sample? The number of molecules depends on the length of the DNA molecule and whether it is single or double-stranded. Assume the identical DNA molecules are single-stranded and 4,000 nucleotides long. The average molecular weight of a nucleotide is 300 grams/mole.
b. A sequencing reaction has a ratio of 300 molecules of ATP to 1 molecule of dATP. What is the probability that termination will occur for a given position in the growing strand when it is opposite a T in the template strand? In other words, what percentage of the time will a dATP be incorporated versus a dATP in the growing strand?
c. Double-stranded DNA will convert to single-stranded DNA if heated high enough; this temperature is known as the DNA melting point. Which DNA sequence would you expect to have a higher DNA melting point, one with 60% GC or 40% GC? Explain your answer based upon the number of hydrogen bonds a GC base pair can form
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