Polymerase Chain Reaction

One round of heating and cooling in PCR = 1 cycle and doubles the amount of DNA. After 3 cycles, how much DNA would you expect if your starting sample had 3pg of target DNA?

24 pg

Which of the following would be advantages and disadvantages of PCR with respect to Western blotting for the detection of gene activation? A. PCR does not rely on the availability of antibodies. B. PCR can only detect very large amounts of DNA in a sample. C. PCR will not give you information about the translation of a gene. D. PCR can only detect protein levels in certain organelles.

???? (A and D)

Primer design is a critical step for detection of viruses in a sample. PCR primers are short segments of DNA (about 20 nucleotides long) complimentary to the DNA that is being amplified. A pair of primers is designed and added to the sample. Which of the following would be true regarding primers/primer design? Choose all that apply. A. PCR primers differ from primers produced in DNA replication within cells since they are composed ofDNA not RNA. B. RNA primase must be added to samples to create primers in a PCR reaction mix. C. The primers must not be complimentary to any regions of human RNA/DNA for detection of viral DNA. D. The viral genome must be known to effectively design primers. E. Primers are optional in PCR if you want to amplify the entire template of DNA, not just specific regions. F. Pairs of primers must be designed, one to start amplification with Taq polymerase and one with human polymerase. G. Pairs of primers must be designed, one for each side of the region of DNA to be amplified within a single DNA strand.

???? (A, D, G)

One application of PCR is DNA fingerprinting where DNA gel band patterns are compared between samples. For example, DNA collected at a crime scene can be compared to that of potential suspects.In this technique, specific regions of DNA are amplified by PCR and then run on a gel (gel electrophoresis) to produce the band pattern. Primers for genetic markers are used, the most common of these are called microsatellite regions with simple sequence repeats (SSRs). These regions have 2-6 nucleotides repeated multiple times and the number of repeats is unique to a particular individual. Typically, multiple genetic markers are compared at once.Gel electrophoresis is used to analyze the products of PCR in DNA fingerprinting. DNA is negatively charged so it migrates toward the positive anode at a rate dependent on its size with the smallest pieces migrating the fastest. Because genetic markers will be different sizes for each individual, each person has a unique band pattern.Select all that apply in regards to this technique. A. Coding regions of genes are the most often targeted regions of DNA for DNA fingerprinting. B. This technique can be used with tiny amounts of crime scene DNA collected. C. Each genetic marker amplified would be represented as a band on a DNA gel. D. The technique of DNA fingerprinting is being replaced by whole genome sequencing. E. Can be used to analyze crime scene DNA. F. Can be used to determine paternity and other familial relationships. G. Is frequently used to determine if particular genes are mutated. H. In the banding pattern on a DNA gel, the smallest DNA fragments can be found toward the bottom of the gel.

???? (B, C, E, F, H)

Magnesium chloride (MgCl2) is also required for amplification of target DNA by PCR. Which of the following is likely a role of Mg+ in this technique? Choose all that apply. A. Acts as a cofactor for polymerase B. Acts as an allosteric inhibitor of polymerase C. Makes the solution hypertonic to the polymerase enzyme D. The positive charge of the Mg+ ions neutralize the negative charges of the phosphate groups of nucleotides for a more efficient reaction E. Allows for easy isolation of the newly synthesized DNA by charge sorting.

A

PCR based techniques can be used to detect viral diseases very soon after infection since it can amplify miniscule amounts of viral based DNA for detection. For example, it is often used to detect infection of exposed health care workers to HIV soon after the exposure event. The standard HIV test is for antibodies against HIV in blood and requires an acquired immune response that can take weeks to months. What technique could be used to detect antibodies against the HIV virus in a sample? A. PCR B. Blood cell fractionation and centrifugation C. Western Blotting D. Light microscopy of infected cell

A

What allows for the copying (amplification) of particular regions of DNA and not others in PCR? A. The design of primers. B. The types of nucleotides used. C. The type of polymerase used. D. The type of thermocycler used.

A

Speaking of HIV, an important piece of information in the management of a person infected with HIV is his/her "viral load." How could PCR give information on the amount of viral particles (viral replication) in a specific person? Choose all that apply. A. The cycle number of detection is proportional to the starting amount of virus in a sample. B. The higher the cycle number of detection, the greater amount of viral replication. C. A person with a low cycle number of detection would have a relatively high viral load. D. Cycle number of detection and viral load are unrelated.

A and C

What is the purpose of heating DNA in PCR? Select all that apply. A. To kill any contaminating bacteria. B. To separate the DNA strands. C. To denature any contaminating enzymes in the sample. D. To activate the deoxyribonucleotides.

B

Another application of the Polymerase Chain Reaction (PCR) is to screen samples for the presence of viruses. For example, remember the recent Ebola outbreak? In the US, if a patient is suspected of having clinical symptoms and is at high risk for exposure to Ebola virus, one technique to determine if that person is infected with Ebola is to send a blood sample for PCR based analysis. In this assay, many copies of specific DNA sequences are made in order to cross a detection threshold.Each Ebola virus is composed of single strand of RNA approximately 18,950 nucleotides long enclosed within a cylindrical viral envelope. In some laboratories, the first step might be to incubate samples with the enzyme Reverse Transcriptase. Why might this be? Choose all that apply. A. The polymerase chain reaction is identical to all transcription reactions. B. DNA is often the starting material for PCR so the viral genome is first transcribed into RNA. C. DNA is often the starting material for PCR so the viral genome is first reverse transcribed into DNA. D. In PCR, RNA primers bind to specific regions of DNA not RNA so the RNA must first be converted to DNA.

C

PCR utilizes specific DNA primers and a thermo stable polymerase enzyme to amplify (make many copies of) a particular DNA sequence (typically 100-600 bases long). Double stranded DNA (dsDNA) must first be separated and primers anneal (complimentary base pair to) their targeted sequences. Polymerase can then extend the DNA strands. What must also be added to the mixture besides primers and polymerase enzymes in order to have amplification of DNA? Choose all that apply. A. ATP B. RNA polymerase C. dNTPs D. electron carriers E. DNA repair enzymes

C

What is the overall purpose of the Polymerase Chain Reaction? A. To repair damaged DNA B. To make many copies of entire chromosomes C. To make copies of specific regions of DNA D. To prepare cells for cell division

C

Another application of PCR is to determine whether or not a particular gene is expressed. DNA is housed in the nucleus of cells and certain genes can get activated in response to environmental stimuli. When a gene is activated, it is transcribed (RNA is produced). In the case of protein-coding genes, after transcription of the gene (production of mRNA), translation often occurs resulting in the production of a particular protein.To determine if a particular gene has been expressed, all RNA from a cell sample is collected and complimentary DNA (cDNA) is created by incubating the RNA with reverse transcriptase. Primers are then designed for the gene of interest. Can a researcher just collect all of the DNA in a cell sample and use it instead? A. Yes, the outcome will be identical. B. Yes, but several genes may get amplified at once. C. No, PCR does not work with DNA as a starting material. D. No, it will not answer the question of whether a particular gene has been activated and transcribed but will instead amplify genomic DNA.

D

The polymerase used in PCR is called Taq polymerase and is isolated from the hot pool bacteria, Thermus aquaticus. Why would human polymerase not be suitable for PCR based methods to determine if infection with Ebola had occured? Choose all that apply. A. Human polymerase would not recognize viral DNA. B. It is unethical to harvest human polymerase. C. Human polymerase is much larger and more complicated as compared to bacterial polymerase. D. Human polymerase would denature under the conditions of PCR. E. Human polymerase would get infected by Ebola DNA and not catalyze the reaction.

D

With regards to the previous question, how would PCR give different information than western blotting about whether a particular gene is expressed? A. PCR will tell you if the gene was transcribed. B. PCR will tell you if a protein product was made. C. Western blotting will tell you if a gene was transcribed. D. Western blotting would tell you if a protein product was made.

D

Standard PCR amplifies specific target regions of DNA. RT-qPCR (quantitative real time PCR) incorporates internal standards and flourescent probes that insert in (intercalcate) with double stranded DNA. This allows for the detection and quantification of PCR products (see simplified diagram).It usually takes many cycles before viral derived DNA can be detected. A lab is analyzing two samples from possible Ebola patients. One sample (S1) detects viral derived DNA at cycle 35 and the second sample (S2) detects viral derived DNA at cycle 23. Which sample S1 or S2 has higher levels of viral particles in the starting sample?

S2