BIOC2000 Lecture Notes - Lecture 9: Polymerase Chain Reaction, Reverse Transcriptase, Antiviral Drug

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ceriseelk223

21 May 2018

School

Department

Biochemistry

Course

BIOC2000

Professor

Dr Justin Ridge

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Lecture 9: PCR

Elimination of the 3’ end as the mode of action for a potent

antiviral drug; AZT acts as a chain terminator

• 3’OH can be used in drug design

• AZT is anti-HIV drug

• AZT is taken up by cells where it is converted into triphosphate. The

reverse transcriptase of HIV prefers AZT triphosphate to the normal

nucleotide dTTP. A reverse transcriptase (RT) is an enzyme used to

generate complementary DNA from an RNA template, a process

termed reverse transcription - associated with retroviruses

• dTTP and AZT structure is the same except OH is replaced with NH3

does not have lone pair arrangement like OH, cannot do a

nucleophilic attack, making it a ‘chain terminator’ – can be added to

DNA change, cannot continue the chain

What does DNA Polymerase need to function?

1. Template

There must be a template strand to be copied.

2. Primer

DNA pol I (and every other known DNA polymerase) cannot initiate

DNA synthesis by itself. It can only extend a pre-existing DNA chain.

3. 3’ OH end

The reaction mechanism requires that the primer must have a free

3'OH end for synthesis to continue

4. dNTPs

Restriction enzymes are required later to process DNA so it can be cloned.

Polymerase chain reaction (PCR) is a technique used to amplify a single

copy or a few copies of a segment of DNA across several orders of

magnitude, generating thousands to millions of copies of a particular DNA

sequence.

Given a piece of ds DNA, running antiparallel (5’-3’, 5’-3’ backward). We

want to replicate this piece of DNA using PCR. Primers need to be built that

will allow us to bind to DNA and extend.

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Document Summary

The reverse transcriptase of hiv prefers azt triphosphate to the normal nucleotide dttp. Dna change, cannot continue the chain lone pair arrangement. What does dna polymerase need to function: template. There must be a template strand to be copied: primer. Dna pol i (and every other known dna polymerase) cannot initiate. It can only extend a pre-existing dna chain: 3" oh end. The reaction mechanism requires that the primer must have a free. 3"oh end for synthesis to continue: dntps. Restriction enzymes are required later to process dna so it can be cloned. Polymerase chain reaction (pcr) is a technique used to amplify a single copy or a few copies of a segment of dna across several orders of magnitude, generating thousands to millions of copies of a particular dna sequence. Given a piece of ds dna, running antiparallel (5"-3", 5"-3" backward). We want to replicate this piece of dna using pcr.

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

Multiple answers to each question might be possible!

You decide to identify the CFTR mutation by analyzing the genomic DNA of your patients compared to healthy individuals. You specifically are looking to see whether a specific 3' gene truncation has occurred in the patients. You will determine this using hybridization techniques with samples from healthy and CF patients. Which of the following will allow you to accomplish this?

	Using an RNA probe complementary to the region not removed by the truncation.
	Using an RNA probe complementary to the region removed by the truncation.
	Using an DNA probe complementary to the region not removed by the truncation.
	Using an DNA probe complementary to the region removed by the truncation.

To conduct the hybridization experiment, you are trying to decide between using a DNA or RNA probe. Which would be ideal to use and why?

	As both are composed of nucleic acids, using either would result in identical results.
	An RNA probe because RNA has uracil bases.
	An RNA probe because it could also be used in a translation experiment.
	A DNA probe because it is more stable than RNA.
	A DNA probe because RNA cannot bind to DNA.

One step of the Hershey/Chase experiment involved blending the virus/cell mixture before centrifugation and probing the pellet for radioactivity. Why was the blending step necessary?

	To collect the bacteria at the bottom of the tube.
	To break open the bacteria to release the genome.
	To separate the bacteria from the bacteriophages.
	To be able to detect the radioactivity.

Imagine Hershey/Chase had used an RNA virus (genome composed of RNA) instead of a DNA virus in their experiment. Would radioactivity still have been found in the pellet?

	No, because only DNA can be labeled with radioactivity.
	No, because the RNA genome would not enter the bacteria upon infection.
	No, because while DNA and RNA nucleotides are similar, they are not identical.
	Yes, because DNA and RNA nucleotides are similar.
	Yes, because genome in any form (DNA, RNA, protein) would be labeled similarly.

The human genome consists mostly of non-coding DNA. Which of the following are benefits of this?

	Random DNA mutations generally won't affect RNA and protein function.
	It is faster to duplicate the genome when these are present.
	The existence of introns can lead to multiple variations of proteins encoded by a single gene.
	It is unlikely transposons would exist in the genome if there was too much protein coding DNA.

Explain the 5â to 3â directionality of a DNA stand.

	It is due to the fact that the free 5â carbon is on one end and the free 3â carbon is on the other
	It is due to the fact that new nucleotide are added to the 5â carbon of the previous nucleotide
	It is due to the fact that there are 3 phosphate groups attached to the 5â carbon
	It is due to the fact the complementary strand is 3â to 5â
	More than one of the above explain the 5â to 3â directionality

You accidentally add a mutant dNTP (which has an H instead of an OH connected to the 3â carbon) to a reaction where DNA is being replicated. Which of the following is true of this mutant dNTP?

	It can be incorporated into DNA strand but cannot form a phosphodiester bond with an incoming wild type dNTP
	It can be incorporated into a DNA strand but cannot base pair with a complementary nucleotide
	It can be incorporated into a DNA strand and can form a phosphodiester bond with an incoming dNTP, but only if it is another mutant dNTP
	It cannot be incorporated into a DNA strand.

Why does DNA polymerase utilize an RNA primer?

	DNA polymerase is unable to initiate strand synthesis but RNA polymerase can
	DNA polymerase can only add a dNTP to an rNTP
	DNA synthesis proceeds in the 3â to 5â when initiating strand synthesis
	Chromosomal DNA contains interspersed RNA fragments
	The RNA primer increases stability of the newly synthesized strand

rosehippopotamus950

Class 6: Viruses (and Cells, continued)

Making connections: viruses and microbes Match the cell biology concept (on page 1) with an important application of that concept (on page 2). Then answer the follow-up questions at the bottom of page 2.

Cell Biology concepts
1. Bacteria that cannot construct new cell wall material become fragile and break down.
2. During influenza infection, the viral enzyme neuraminidase is required to release new viruses from the host cell in which they were made. Once released, the new viruses can infect many additional cells.
3. Thermus aquaticus is a bacterium found in hot springs and can live at temperatures up to ~80Â°C (175Â°F). It has adapted so that its enzymes are most functional at the elevated temperatures, compared with many organismsâ enzymes that function best ~37Â°C (98Â°F).
4. Until the isolation and investigation of retroviruses, scientists thought that RNA could be copied from DNA, but DNA could not be copied from RNA. Research on this class of viruses led to an update to this âCentral Dogmaâ of biology.
5. Some bacteria have adapted, so that the enzymes involved in making cell wall materials have a slightly different shape and can no longer be bound by certain drugs.
6. Rhizobia are bacteria that colonize the roots of certain plants (legumes, including beans and peas). The host plant benefits from the prokaryotesâ ability to âfixâ nitrogen, converting N2 from the atmosphere into a form that is usable by the plant.
7. Many species of bacteria contain the enzyme cellulase. More than 10 billion such bacteria and other symbiotic microbes may be found in a single milliliter of material from inside the digestive system of ruminant mammals.
8. Fungi, including those that cause athleteâs foot, use ergosterol to maintain healthy membranes. Animal cells do not make ergosterol, they use a similar lipid â cholesterol.
9. During retrovirus infection, a DNA copy is made of the virusâs RNA genome. A cut is made in the host cellâs DNA and the DNA copy of the viral genome is pasted into the host chromosome by a viral enzyme called integrase.
10. Prokaryotic cells contain smaller ribosomes (â70Sâ ribosomes) than eukaryotic cells (â80Sâ ribosomes). Both types of ribosomes synthesize proteins, but the macromolecules they use to do it are somewhat different.

2
Applications
_______ Oseltamivir, also called Tamiflu, binds and inhibits the viral protein neuraminidase.
_______ Clotrimazole is a drug that is used to treat certain infections. It interferes with synthesis of ergosterol, a lipid involved in phospholipid membranes.
_______ Penicillin is a drug that interferes with synthesis of peptidoglycans (these are biological molecules that are part protein and part carbohydrate) that are present in certain cell walls.
_______ HIV is often treated with several drugs that inhibit different parts of the viral replicative cycle. Raltegravir is an example of a drug that interferes with the integration of viral genes into the host cellâs chromosome.
_______ Cows, like other vertebrates, lack the ability to breakdown cellulose. Yet they are able to live on a diet of grass and other vegetation high in cellulose.
_______ Enzymes isolated from extremophiles have been very useful in laboratory research. For example, Taq polymerase facilitates PCR, a revolutionary technique for production of DNAs of particular sequences, which requires cycling reactions through high temperatures.
_______ Plants and animals require nitrogen to make biological macromolecules, however theyâre unable to convert atmospheric nitrogen (N2) into a biologically useful form.
_______ Azithromycin is a drug that binds to 70S ribosomes and blocks synthesis of any new proteins.
_______ Methicillin-Resistant Staphylococcus aureus (MRSA) can cause life-threatening infections of the skin and bloodstream. One of the reasons MRSA infections are so serious is that they are resistant to treatment by many antibiotics, including methicillin and penicillin.
_______ The enzyme reverse transcriptase, isolated from retroviruses, is an important tool in molecular biology research. Scientists interested in learning what genes are turned on in particular cells use it to make DNA copies of RNAs they are studying (The DNA copies are useful because they are more stable).

When you have completed the matching activity, answer these follow-up questions:
â¢ What types of infections can be treated with the following drugs?
o Penicillin
o Clotrimazole
o Azithromycin
o Tamiflu
o Raltegravir
â¢ Why are these drugs (above) safe for you to take? That is, why donât they kill your cells?
â¢ Cancer cells are your own human cells that have become deregulated so that they divide and pile up in an uncontrolled way. Many chemotherapy drugs treat cancer by interfering with cell division and other cell processes.
o Why are there so many more side effects for individuals undergoing chemotherapy than for individuals taking antibiotics?

BIOC2000 Lecture Notes - Lecture 9: Polymerase Chain Reaction, Reverse Transcriptase, Antiviral Drug

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