Chapter 4 Homework

2. Suggest two different strategies for synthesizing a 0.5kb gene. Discuss the
advantages and disadvantages of both methods
The first strategy that comes to mind after reading chapter 4 is to use the
chemical synthesis of DNA process. This would involve creating DNA from
scratch using CPG beads and numerous coupling reactions. However the
chances for creating the 0.5kb gene from the coupling reactions alone would
be slim to none as the percent of desired product drops after each
oligonucleotide is added. So I would instead synthesize complimentary
fractions of the larger gene that had overlapping ends that would anneal one
another. Once combined you would use T4 ligase to repair the nicks.
The second strategy that might be used would be to conduct a PCR reaction.
Using double stranded overlapping oligonucleotides begin the PCR process.
After the ligation step, DNA polymerase will fill in the blanks. Proceed with
this reaction until there is enough sample. Add a second set of
oligonucleotides and repeat until the full gene has been synthesized.

3. What is a linker? How is it used?
Linkers are short double stranded DNA molecules that are blunt ended,
contain a restriction endonuclease site, and are self-complementary. They
are commonly ligated to DNA molecules lacking restriction sites, allowing
them to be cloned.

4. What are the essential components of a PCR used to amplify a specific
sequence of DNA?
The essential components of a standard PCR would include: gDNA or cDNA
or even a synthetic variant, a set of primers, dNTPs, buffer solution, DNA
polymerase (usually taq polymerase), and most likely diluted to a specific
concentration with H2O. The genomic DNA acts as a template strand from
which subsequent strands are copied. The primers are short sequences ~20
that flank either side of a complementary sequence. The
deoxyribonucleotides (dNTPs) are the A, T, G, and C building blocks of the
DNA. The DNA polymerase should be able to withstand temperatures around
95°C, typically taken from thermophilic Achaea. The buffer solution is meant
primarily for the DNA polymerase, which may be denatured under
suboptimal pH conditions.

5. Outline the steps in a PCR cycle.
a. Denature the template sequence as fully as possible at ~95°C
b. Drop the temperature to allow the primer sequences to anneal.
The temperature for this is very particular and should be
calculated based on the G,C to A,T ratio, but typically between 5060°C.
c. Raise the temperature to ~74°C. This allows, in this case, taq
polymerase to catalyze the synthesize DNA at it’s optimum.
d. Repeat the above steps until you have your desired amount of
DNA. The amount of starting DNA should grow exponentially after
each round of denaturation, renaturation, and synthesis.

8. What is a primer? What are the key requirements of an effective primer?
Describe some of the techniques that require primers.
A primer is a ~20bp strand of DNA that binds to a complementary sequence
within a larger template. Primers should be complementary to a sequence
within one gene and one gene alone, so as not to amplify an undesired
sequence. PCR amplification requires primers as does DNA-sequencing as
does cDNA synthesis as does the random primer labeling method.

9. What is a dideoxynucleotide? How is it used to determine the sequence of a
DNA molecule?
A dideoxynucleotide is an artificial molecule with some semblance to DNA
but with 2’ and 3’ carbons missing their hydroxyl groups. The unique feature
of this molecule is that they inhibit DNA synthesis when they are added to a
DNA molecule. To determine a DNA sequence one would prepare four tubes
of radiolabeled deoxynucleic acid along with a precise concentration of
didoxynucleotides, so that they are added to the growing DNA strands at
each pase pair of their growth. This gives you a pool of DNA of numerous
lengths. Using a polyacrylimade gel, which can differentiate between up to
1bp, separate the DNA and observe the length and base pair type of each
radioactively labeled strand. This forms a long map, which tells us our DNA
sequence.

10. Why is it necessary to make DNA single stranded before determining its
sequence?
This is required in order for the single stranded DNA sequencing primer to
bind/hybridized to one of the DNA template strands.

11. Draw the autoradiograph derived from dideoxynucleotide sequencing of
CCTGATCTTAGCCAT.
ddATP
ddCTP
ddGTP
ddTTP
3’
T
A
C
C
G
A
T
T
C
T
A
G
T
C
C
5’

12. Outline the basic features of pyrosequencing.
Pyrosequencing is a sequencing method in which the release of
pyrophosphate is detected when a known nucleotide is added to a growing
complementary DNA strand. Pyrophosphate is produced only when the
proper complementary base is added. The pyrophosphate triggers a reaction
involving the enzymes ATP sulfurylase and luciferase to produce detectable
light. The light can then be used to determine how many bases have been
added. The light emission is used to generate a pyrogram that can reveal the
sequence of the strand.

13. How are incorporated nucleotides recognized after each cycle of
sequencing by synthesis using reversible chain terminators? How does this
differ from pyrosequencing?
In pyrosequencing each nucleotide must be added separately. Reversible
chain terminators and four color fluorescence have made it possible to speed
this process by combining all four nucleotides in one reaction. One approach
involves the use of an agent to block the 3’ hydroxyl end and a fluorescent
dye that is unique to each nucleotide. Currently these techniques are limited
to short sequences due to limitations with cleavage of the fluorescent dye
and blocking agent.

16. What are the advantages of cycle array sequencing compared to the
shotgun cloning method for large-scale sequencing?
Cycle array sequencing is a much more effective technique than shotgun
sequencing. Shotgun sequencing has the issue of false contigs or repetitive
elements that actually occupy a different place in the genome but are so
similar that they are assigned the same location. In addition, shotgun
sequencing is time consuming and much more costly than cycle array
sequencing. Cycle array sequencing also has the benefits of using less
reagent and allowing libraries to be prepared in vitro. With this method it is
also possible to avoid having to prepare clone libraries in bacterial cells
because the reagents are simply attached to the surface of beads..