BIOLOGY 172 Lecture Notes - Lecture 21: Dideoxynucleotide, Transposable Element, Dna Profiling

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15 Oct 2016

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When we look at our dna, we want to see the nucleotides. One of the ways to find the sequence of dna is by the use of dideoxynucleotides. Dideoxynucleotides has a missing o on the 3rd prime carbon. When dna polymerase encounters this, the dna will stop dna synthesis. All four bases of dideoxynucleotides are added to the solution and are colored. First you have the template strand that you want to know the sequence off. You have to start off with a primer to start the sequencing. The primer can start with either dideoxynucleotides or the regular nucleotides (deoxynucleotides). Dideoxynucleotides will come to a halt since dna polymerase cannot keep on extending. This process will continue and the end result will be a range of products with a different color that will have a different dideoxynucleotides. During this process, the dna polymerase can choose between the regular base or the di-base.

Related Questions

Please answer all

Why does a template-dependent DNA polymerase require a primer to initiate DNA synthesis?

	DNA polymerase require 5' phosphate to add a new nucleotide
	DNA polymerase require 3' hydroxyl group to add a new nucleotide

DNA polymerase require energy by hydrolizing the primers

Which of the following enzymes is template independent?

	Reverse transcriptase
	Klenow fragment

	DNA polymerase
	Terminal deoxynucleotidyl transferase

RNA polymerase

The natural function of a DNA ligase enzyme is:

	Joining together DNA sequences with compatible cohesive ends from two different organisms to yield a recombinant DNA molecule
	Sealing single stranded nicks in double stranded DNA molecules

	Joining together two blunt ended fragments of DNA within a cell
	Joining together two single stranded DNA molecules

The natural function of 3' -> 5' exonuclease activity of a DNA polymerase is to:

	Remove the 5' end of the DNA strand that is being copied
	Remove damaged nucleotide from the template strand

	Remove nucleotides from the end of DNA to generate blunt ends
	Remove incorrect nucleotides from the newly synthesized DNA

Which technique is used to resolve the different sizes of DNA fragments?

	Gene cloning
	PCR

	DNA sequencing
	Gel electrophoresis

Which of the following vectors does not contain bacterial origin of replication (oriC)?

	plasmid
	cosmid

	bacterial artificial chromosome
	lambda vector

Which process of bacteriophage is not used for gene cloning?

	Concatemer formation
	Lytic cycle

	Lysogenic cycle
	viral packaging

What vector would be best suited for creating a contig of bovine (cattle) chromosome 10?

	lambda vector
	Cosmid vector

	P1 vector
	Plasmid

E. coli takes up plasmid DNA by which of the following methods?

	Transduction
	Transformation

	Conjugation
	Transfection

The following times and temperatures are an example of the steps for PCR. You can use the Figure to help you answer the following questions.

Why is the first step is carried out at 94Â°C?

	To degrade the template
	To denature the template

	To activate the polymerase
	To facilitate the primer binding

What happens in the reaction when the temperature shifts to 55Â°C during cycling?

	the DNA polymerase will carry out DNA synthesis by extending the annealed primers
	the DNA polymerase will finish DNA synthesis

	the primers anneal to the single-stranded regions of the DNA
	the primers anneal to the double-stranded regions of the DNA

During cycling, what occurs when the temperature is at 72Â°C?

	the primers anneal to the double-stranded regions of the DNA
	the DNA polymerase will carry out DNA synthesis by extending the annealed primers

	the primers anneal to the single-stranded regions of the DNA
	the DNA polymerase will finish DNA synthesis

You designed a set of primers to amplify 1 kb DNA with polymerase chain reaction. By which cycle of polymerase chain reaction, we would expect to see the first double stranded DNA with expected size?

	The end of cycle 1
	The end of cycle 2

	The end of cycle3
	The end of cycle 4

Multiply questions here

Q2. What is the consequence of chromosome inversion on meiosis? (work on a zygote with one normal chromosome and one chromosome with an inversion)

Q3. Search the web or read the textbook. Find a disease that is caused by translocation. What kind of translocation causes the disease?

Q4. Briefly explain how downâs syndrome or other trisomy is generated (3 sentences should be enough for you to explain this clearly)

Q5. I mutated a culture of wild type E. coli cells and wanted to isolate mutants that are unable to synthesize uracil (a nitrogen base in RNA). How can I achieve my goal? (try to finish your answer in 2-3 sentences)

Q6. For the ura- mutant isolated in Q5, I want to convert it to URA+ by transformation. Please tell me how I can achive this transformation with 3 SIMPLE sentences

Q7. What is the difference and relationship between an Hfr cell and an F+ cell?

Q8. I want to set up a mating between an Hfr strain and an F- (ade- his- bio-) strain and detect bio+ recombinant. Please design the experiment for me. (What genotype for Hfr do you need to choose? How will you select your recombinant? etc).

Q9. I want to use phage T2 to transducer the wild type ADE2 gene from the wild type E. coli strain to an ade2- E. coli mutant. How can I do this experimentally? (please briefly describe the approach, not experimental details such as incubation for 10 min, etc).

Q10. We talked about recombination in both eukaryotes and prokaryotes. Are there any differences in these two recombination ? (what are they if there are?)

Q11. Whose (or what) experiment proves that DNA is the genetic material?

Q12. What knowledge did Watson and Crick use in constructing the DNA model?

Q13. How does the double helix model support the three major functions of genetic materials?

Q14. What is the major chemical force that holds DNA and histone together? Hydrogen bonds

Q15. If I label E. coli cells in an N15 medium overnight and chase cells in an N14 medium for 2 generations, what fraction of cells still carry the N15 label?

Q16. If a 1000 kilobase fragment of DNA has five evenly spaced replication origins and DNA polymerase moves at 1 kilobase per second, how many seconds will a cell take to produce two daughter molecules?

Q17. DNA polymerases catalyze the polymerization of nucleotides. Use the formula for enzyme catalyzed reaction (see the example) to describe DNA polymerases. Example: Enzyme Substrate A + substrate B -------------------ï product c + product D Q8. During DNA replication, what is the direction of the growing strand?

Q18. For DNA synthesis in cells, why are there a leading strand and a lagging strand?

Q19. The following is the DNA sequence of a template strand. If the primase begins to synthesize primers from the middle âgâ, what will the primer sequence be? (assuming the primer can be 8 nucleotides long) 5â GACTCTAgGACTCCT 3â

Q20. Both DNA polymerase I and DNA polymerase III synthesize DNA during DNA replication. Please state the different roles they played during DNA replication.

sangriawallaby972

Answered most of the questions. Can you tell me if my answers are correct? Last question of the month. Thank you. I appreciate it.

4. You perform electrophoresis on the product of a PCR and see that instead of the 300-base band you expected that there are five different bands.

What controls the specific sequence to which a PCR primer binds? Complementary sequence and annealing temperature control the specific sequence to which a PCR primer binds.

What are two factors that contribute to the annealing temperature of the primer? Two factors that contribute to the annealing temperature of the primer is primer length and the Guanine and Cytosine content.

What part of your thermal cycle protocol could you change, and how would you change it, to try and get rid of the extra bands?

To get rid of the extra band, we have to increase the annealing temperature.

If, after performing electrophoresis, there were no bands on the gel, what part of your thermal cycle protocol would you change, and how would you change it?

After performing electrophoresis, if there were no bands on the gel then we have to decrease the annealing temperature of the primer

5. If you are using a DNA sequence from a related species to design primers for your species of interest, why is it better to design your primers to complement an exon?

In related species there are similarities but their genetic makeup will not be the same. It is better to design your primers to complement an exon because ??

6. Why is it better to use a 25-base primer than a 10-base primer? Why do you have to have a lower annealing temperature for a 10-base primer?

It is better to use a 25-base primer than a 10-base primer because a 10-base primer is not long enough and may pair with a wrong portion of DNA, which would give wrong results. You have a lower annealing temperature for a 10-base primer because annealing temperature increases when the number of base pairs increase. If the temperature is too high the primer might not bind.

Identify the three steps in PCR and tell the function of each step.

Denaturation - when the double-stranded DNA is heated to 92-95â for about one minute, to separate it into two single strands.

Annealing - temperature of the reaction is lowered to a temperature that is between 45â and 65â to allow the DNA primers to attach to the template DNA.

Extension - temperature of the reaction is raised to a temperature between 45â and 65â and the new strand of DNA is made by adding nucleotides to the ends of the primers.

BIOLOGY 172 Lecture Notes - Lecture 21: Dideoxynucleotide, Transposable Element, Dna Profiling

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