Unit 4 PCR

B. To use DNA as the starting material of PCR so the viral genome is first reverse transcribed into DNA

Another application of the Polymerase Chain Reaction (PCR) is to screen samples for the presence of viruses. For example, do you remember the Ebola outbreak? A blood sample can be sent for PCR based analysis. In this instance, many copies of viral specific DNA sequences are made to cross a detection threshold. Each Ebola virus is composed of a single strand of RNA approximately 18,950 nucleotides long enclosed in a cylindrical viral envelope. In some laboratories, the first step might be to incubate the samples with the enzyme reverse transcriptase. Why might this be? A. To use DNA as the starting material of PCR so the viral genome is first transcribed to RNA with reverse transcriptase B. To use DNA as the starting material of PCR so the viral genome is first reverse transcribed into DNA C. DNA is more reactive than RNA so less energy has to put into the system D. Primers can only be designed with uracil and not thymine

A. Antibodies are proteins with specific three dimensional shapes critical to their function

Antibodies specific to HIV can only bind and inactivate particular strains of the HIV virus and if the virus mutates, the antibodies against the original strain may no longer be effective. Why might this be the case? A. Antibodies are proteins with specific three dimensional shapes critical to their function B. Mutated viruses can deactivate antibodies C. Antibodies might denature under conditions resulting from infection with the new viral strain D. Antibodies may no longer be able to catalyze the reaction that destroys the virus

4 fold more concentrated in 23 than 25

How does the Ct relate to the amount of input targets? If the Ct for Sample 1 is n=23 and Ct Sample 2 is n=25 , what is the relative difference in target DNA concentrations for the sample inputs?

Design primers are only going to bind to the target

How is target specificity achieved with primer design?

Detected at lower rates. How well it detects

How would you define analytical sensitivity and specificity for PCR?

A. 3.2 x 104

If before PCR, a patient had a viral load of 1.6 x 104 viral particles. What would be the amount of DNA in the sample after the completion of the first PCR cycle? A. 3.2 x 104 B. 1.6 x 108 C. 3.2 x 1016 D. 1.6 x 1032

B. Approximately 8 times more

If it can be assumed that viral detection occurred with the same amount of DNA in S1 and S2 (S1 detected at cycle 35, S2 at cycle 32) - How much more viral derived DNA was in the starting sample of S1 or S2? A. Twice as much B. Approximately 8 times more C. Approximately 16 times more D. Impossible to determine

D. 3000 Mb

If the human genome is approximately 3.0 x 109 base pairs , how many mega (million) base pairs (Mb) is the human genome? A. 3 Mb B. 30 Mb C. 300 Mb D. 3000 Mb

C. C

Looking at the attached gel image, which band is approximately 1000bp? A. A B. B C. C D. D

A. A

Looking at the attached gel image, which band is approximately 500bp in size? A. A B. B C. C D. D

A. Act as a cofactor for polymerase

Magnesium Chloride (MgCl2) is also required for making copies of target DNA by PCR. Which of the following is the most likely role of Mg+ in this technique? A. Act as a cofactor for polymerase B. Act as an allosteric inhibitor of polymerase C. Makes the PCR solution hyperosmotic to the polymerase enzyme D. Allows for easy isolation of newly synthesized DNA by charge sorting

C. Identical twins

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 separated on an agarose gel by a technique called gel electrophoresis. The specific regions, called genetic markers, are often microsatellite regions of 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. Since genetic markers vary between individuals, each person will have a unique band pattern. How are DNA fragments (that are different sizes in different people) separated onto an agarose gel by gel electrophoresis? DNA is negatively charged and migrates toward a positive anode with an applied electrical current at a rate dependent on its size. The smallest fragments migrate the fastest in the gel. Which of the following pairs of individuals would you expect to have the most similar DNA fingerprints? A. Siblings within a family B. Parent and child C. Identical twins D. All of the above would be equally similar

D. 24pg

One cycle of PCR (one heating and cooling) doubles the amount of DNA. After three cycles, how much DNA would you expect if your starting sample had 3pg of target DNA? A. 6pg B. 9pg C. 12pg D. 24pg

B. 5' CCGACGACCGTTGAA 3'

One of the primers that may be used in the detection of a specific sequence of DNA is: 3' GGCTGCTGGCAACTT 5 Which sequence would be targeted by this primer? A. 5' GGCTGCTGGCAACTT 3' B. 5' CCGACGACCGTTGAA 3' C. 5' TATAACCGTCCAAGTT 3' D. 3' CCGGCCTAGCATAGAA 5'

B. Transfer RNA

PCR can also be used to determine if a particular gene has been expressed (transcribed) in a cell. DNA is housed in the nucleus of cells and certain genes are activated in response to the environment. When activated, mRNA corresponding to the gene is produced. In the case of protein-coding genes, after the production of mRNA, translation often occurs and a protein is produced. To determine what genes have been expressed (transcribed into mRNA), 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. Besides mRNA, what may also end up as part of the cDNA collected from human cells? A. Genomic DNA B. Transfer RNA C. Ribosomal subunits D. Prokaryotic RNA

B. Rough endoplasmic reticulum

PCR techniques can be used to detect viral diseases very soon after infection since miniscule amounts of viral derived RNA/DNA can be amplified for detection. For example, it is often used to detect infection of exposed health care workers to viruses such as hepatitis and HIV soon after the exposure event. The standard HIV test is for antibodies against HIV in circulating in blood which requires an acquired immune response that can take weeks to months. In what cellular organelle would the protein antibodies against HIV be produced? A. Lysosomes B. Rough endoplasmic reticulum C. Mitochondria D. Golgi apparatus

B. dNTPs

PCR utilizes specific DNA primers and a polymerase enzyme to make copies of a particular DNA sequence. The primers must attach (anneal) to separated DNA at the target sequences so that polymerase can build new DNA strands. What must also be added to the mixture besides primers and polymerase enzyme in order to get amplification of DNA? A. RNA polymerase B. dNTPs C. electron carriers D. DNA repair enzymes

B. If detecting viral infection by PCR, the primers must not be complimentary (match) to regions of human DNA

Primer design is a critical step for the detection of a particular sequence in a sample. Which of the following would be true of primer design? A. RNA polymerase are added to the PCR mix to design random primers B. If detecting viral infection by PCR, the primers must not be complimentary (match) to regions of human DNA C. Pairs of primers must be designed, one to start amplification with Taq polymerase, the other to start amplification with human polymerase D. Primers are optional if you want to amplify all DNA within a sample

C. A person with a low cycle number of detection would have a relatively high viral load

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 about the amount of viral particles (viral replication) in a specific person? A. The cycle number of detection is proportional to levels of circulating antibodies B. The higher the cycle number of detection, the greater the amount of viral replication C. A person with a low cycle number of detection would have a relatively high viral load D. Cycle of detection and viral load are unrelated

B. Replication

The Polymerase Chain Reaction (PCR) is most comparable to what cellular process? A. Mitosis B. Replication C. Transcription D. Translation

C. S3

The attached image is a gel of crime scene and suspect DNA. Which suspect is most likely the perpetrator of the crime? LC=loading control CS=crime scene sample S1-S4=suspects 1-4 A. S1 B. S2 C. S3 D. S4

C. 150, 000 times larger

The ebola viral genome is approximately 18,950 nucleotides. The human genome is approximately 3.0 X 109 base pairs. Approximately how much larger is the human genome as compared to the viral genome? A. 15 times larger B. 150 times larger C. 150, 000 times larger D. 1.5 million times larger

C. Human polymerase denatures under conditions of PCR

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? A. It is unethical to harvest human polymerase B. Human polymerase are more complicated and difficult to work with C. Human polymerase denatures under conditions of PCR D. Only Taq polymerase can recognize viral DNA if using for viral detection

A. specific primers used

What allows for the amplification (making of many copies) of particular regions of DNA but not others in PCR? The .... A. specific primers used B. types of nucleotides used C. types of polymerase used D. type of thermocycler used

In contrast, conventional DNA typing needs at least 50 to 500 ng of high molecular weight DNA, and in most cases repeat analysis. The use of PCR for the analysis of DNA from evidence samples has also made it possible to use other material which could connect certain subjects with a crime.

What is the advantage of PCR over conventional DNA methods for ancient specimens, forensic samples or other very rare sources of DNA?

C. To make copies of specific regions of DNA

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

C. dNTPs

What provides the energy for synthesizing DNA strands in PCR? A. ATP added to the PCR mix B. A charge current C. dNTPs D. charge repulsion

D. No, it will not answer the question of whether a gene has been expressed

When creating a sample to look for expression of certain genes by PCR, can the researcher just collect all DNA in the cell sample instead of creating cDNA? A. Yes, the outcome will be identical B. Yes, but several genes will get amplified at once C. No, PCR will not work on genomic DNA D. No, it will not answer the question of whether a gene has been expressed

GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer

When designing primers, why is it critically important to know the end sequences, but less important to know the internal DNA sequence?

C. 3' hydroxyl of deoxyribose

When enzymes are elongating (building) a newly synthesized DNA strand in PCR, new nucleotides are being added to the ____ of the growing strand. A. 5' phosphate group B. A nitrogenous base C. 3' hydroxyl of deoxyribose D. 2' H of deoxyribose

A. Those with a large percentage of G-C base pairs

Which of the following DNA strands might need to be heated to a slightly higher temperature than average in PCR? A. Those with a large percentage of G-C base pairs B. Those with a large percentage of A-T base pairs C. Those with an equal number of G-C and A-T base pairs D. Those with a supercoiled structure

B. This technique requires large amounts of starting DNA

Which of the following is NOT true of DNA fingerprinting? A. Non-coding regions of DNA are most often used as genetic markers B. This technique requires large amounts of starting DNA C. Each genetic marker amplified would be represented as a band on a DNA gel D. Individuals have unique band patterns (aka - fingerprints)

There can be various sources of contamination during PCR, leading to myriad observations that may require troubleshooting. A common observation is excessive or unexpected signal, typically caused by contamination of reagents with template, genomic DNA, or amplicons from previous reactions.

Why is contamination of an amplified product a major problem?

B. Frameshift

Yet another application of PCR is to screen a patient for a particular genetic mutation at a specific gene. For example, there are many genetic mutations that are associated with increased susceptibility to disease. If the genetic mutation being screened for is a result of a single base insertion, what type of mutation would this be classified as? A. Nonsense B. Frameshift C. Missense D. Silent

E. The medicine decreases the expression of the gene but not in a does dependent manner

cDNA is collected and RT-qPCR is run to determine the expression of a particular gene in response to exposure to a new medicine. The untreated sample detection threshold is cycle 5 and treated samples had the following results: Dose 1: cycle 8 Dose 2: cycle 7 Dose 3: cycle 8 Dose 4: cycle 8 What conclusions can the investigators draw from the results? A. The medicine has no effect on the expression of the particular gene. B. The medicine increases the expression of the gene in a dose dependent manner C. The medicine increases the expression of the gene but not in a dose dependent manner D. The medicine decreases the expression of the gene in a dose dependent manner E. The medicine decreases the expression of the gene but not in a does dependent manner

B. S2

tandard PCR amplifies specific target regions of DNA. RT-qPCR (quantitative real time PCR) incorporates internal standards and fluorescent probes that insert within double stranded DNA to allow for the detection and quantification of PCR products. If using RT-qPCR for viral detection, it usually takes many cycles before viral derived DNA can be detected (have to first make many copies with the fluorescent probe to detect). 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 #32. Which sample, S1 or S2, has higher level of viral particles in the starting sample? A. S1 B. S2