What enzyme unwinds DNA ahead of the replication fork?
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Topoisomerase.
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What enzyme unwinds DNA ahead of the replication fork?
Topoisomerase.
What is one key benefit of using PCR in forensic science?
It enables the analysis of degraded or limited DNA samples.
What has DNA analysis become in modern criminal investigations?
A staple.
What does primase do during DNA synthesis?
Generates an RNA primer from which DNAP can initiate the extension process.
What is the challenge faced in studying the HLTH gene?
The gene must be present in high abundance for mutational analysis.
What does PCR stand for?
Polymerase Chain Reaction.
What is the size of the HLTH gene you are studying?
1.5 kb (1500 bp).
How large is the human genome?
~3 billion bp.
What is the purpose of using PCR in this experiment?
To determine if there is Salmonella contamination in poultry samples.
What is the purpose of using PCR in the context of poultry samples?
To determine if there is Salmonella contamination.
What is the Polymerase Chain Reaction (PCR)?
A technique that allows us to synthesize millions (or billions) of copies of a small fragment of DNA.
What is required for DNA synthesis in vivo?
Free deoxynucleoside triphosphates (dNTPs) for each of the 4 bases.
What does it indicate if a band is seen in the negative control lane?
It may indicate contamination or non-specific amplification in the PCR process.
What database can DNA profiles be compared to?
The FBI’s CODIS (Combined DNA Index System).
What is the significance of using a DNA ladder in the gel electrophoresis?
To determine the size of the amplified DNA fragments.
What does PCR stand for?
Polymerase Chain Reaction.
Why is PCR important for matching DNA in criminal investigations?
It increases the quantity of DNA available for comparison.
What were the limitations of forensic analysis before the invention of PCR?
Limited to fingerprinting, blood typing, and obvious physical evidence.
What is the purpose of designing primers in a PCR experiment for Salmonella detection?
To amplify a DNA sequence unique to Salmonella species.
What component of the PCR setup needs to be changed for each sample tested?
The DNA template from the poultry samples.
What would be a good positive control for the PCR experiment?
A sample known to contain Salmonella DNA.
What are the biochemical principles underlying DNA synthesis?
They involve the mechanisms and processes that facilitate the formation of DNA molecules.
What is often part of a larger gel documentation system?
The gel doc.
What are the resulting DNA fragments produced by PCR called?
Amplicons.
What is the primary goal of PCR?
To isolate and amplify a small piece of DNA that we are interested in analyzing.
What type of primers are included in the PCR reaction mix?
Forward and reverse primers (in excess).
What serves as the template in cellular replication?
Cellular DNA.
What is the method of DNA denaturation in cellular replication?
Helicase unwinds the DNA.
What type of primer is used in cellular replication?
RNA primer (primase).
What are the building blocks used by DNA polymerase during DNA synthesis?
Free dNTPs (deoxynucleotide triphosphates).
What serves as the template in PCR?
The DNA sample being amplified.
What type of primer is used in cellular replication?
RNA primer (primase).
What is a key component of the PCR process?
DNA polymerase.
What is the first step in the agarose gel electrophoresis process?
Cast the gel (Optional – apply a pre-stain).
What should be done if a pre-stain was not used?
Stain the gel.
What is generated for a suspect in forensic analysis?
A DNA profile.
What was a major challenge in using DNA for solving crimes before PCR?
Analytical techniques required a larger sample than was usually possible.
How does PCR assist in DNA analysis?
It allows us to multiply a tiny sample of DNA for analysis.
What does the analysis of STRs reveal?
The length of these STRs for that person.
What does a faint band in lane 6 suggest?
It indicates that the sample might contain a small amount of Salmonella, but could also be an error.
What is DNA amplification?
The process that dramatically increases the copy number of a specific sequence of DNA in a sample.
How are complementary nucleotides added during DNA synthesis?
In sequence to the nascent DNA strand (A—T; C—G).
What is agarose gel electrophoresis used for?
It is used to separate DNA molecules based on size.
What is the primary context of PCR?
DNA synthesis in a tube.
Which polymerase is commonly used in PCR reactions?
Taq polymerase.
What ions are typically added to the PCR reaction mix, and why?
MgCl2 is usually added; Mg2+ is an important cofactor for DNA polymerase.
How many errors could be present in the final product after amplification with Taq polymerase?
Over 700,000 errors.
What enzyme is primarily responsible for DNA replication?
DNA polymerase.
What serves as the template in cellular replication?
Cellular DNA.
How do small and large molecules behave in agarose gel?
Small molecules move easily through the gel pores and migrate further, while large molecules get stuck and migrate less far.
What does 'in vivo' mean?
In living cells/organisms.
What is the primary need for PCR in biomedical research?
To isolate and copy specific segments of DNA, such as a gene of interest.
How is stained DNA visualized in agarose gel electrophoresis?
Using UV light to illuminate the stained DNA.
What would be beneficial for studying the HLTH gene in patients?
A method to isolate and copy only the 1.5 kb piece of DNA.
What is a good positive control for the PCR experiment?
A known sample of Salmonella DNA as a template.
What does it indicate if no band is seen in the positive control lane?
It may indicate that the PCR did not work properly or that the Salmonella DNA was not present.
How is stained DNA visualized in agarose gel electrophoresis?
Using UV light.
What would be a good negative control for the PCR experiment?
A sample without any DNA or a sample known not to contain Salmonella.
What is the difference between in vivo and in vitro DNA synthesis?
In vivo occurs within living cells, while in vitro occurs in a controlled environment outside of cells.
How does the gel documentation system record an image of the gel?
It uses a camera with UV filters.
What is the significance of the DNA ladder in the gel image?
It serves as a size reference for the PCR products.
What is the primary function of DNA polymerase in cellular replication?
To extend DNA strands during replication.
What is a quality control application of PCR?
Quality control analysis in consumer products and medications.
What is another application of PCR that requires high fidelity?
Sequencing.
How is PCR used in paternity testing?
To analyze genetic markers for parentage.
What is the first step in DNA replication that involves unwinding the DNA double helix?
DNA denaturation.
What initiates DNA denaturation in cellular replication?
Helicase.
How does agarose gel form?
It polymerizes into a solid gel when dissolved in an aqueous solution.
What is the primary purpose of high-fidelity polymerases?
To create an exact, error-free copy of DNA.
What is the role of DNA polymerase (DNAP) in DNA synthesis?
Adds complementary nucleotides to the growing DNA strand (extension).
What does 'Run to Red' refer to in agarose gel electrophoresis?
It refers to setting up the apparatus correctly.
What role does PCR play in identifying suspects?
It helps in generating DNA profiles from evidence found at crime scenes.
What is the purpose of using PCR in the context of Salmonella contamination in poultry?
To determine if there is Salmonella contamination by amplifying a unique DNA sequence.
In which direction is DNA synthesized?
In the 5’ to 3’ direction.
What type of process is DNA synthesis?
A semi-conservative process.
What should you check when analyzing a gel?
The expected number of DNA fragments, integrity of the sample, length of bands, unexpected bands, and presence of DNA fragments.
What is unique to every individual in DNA profiling?
The DNA profile created from STR analysis.
What should you consider if you do not have a DNA fragment at all?
Your PCR may have failed.
What does the negative control in the PCR experiment help to identify?
It helps to confirm that there is no contamination in the PCR setup.
What is the primary component of the PCR reaction mix that serves as the starting material?
Template DNA.
What type of DNA polymerase is used in PCR?
Thermostable DNA polymerase (Taq polymerase).
What acts as the substrate for DNAP in PCR?
Free deoxynucleotide triphosphates (dNTPs).
How does PCR differ from natural DNA replication?
PCR uses only DNAP and introduces bounds on DNA synthesis.
What is the function of primers in both PCR and cellular replication?
To serve as the starting point of DNA extension.
What is the primary function of DNA polymerase in cellular replication?
To synthesize new DNA strands by adding nucleotides.
What happens when a voltage is applied to the agarose gel?
Opposites attract: negative ions move toward the positive anode and positive ions move toward the negative cathode.
What is PCR primarily used for?
DNA synthesis in a tube.
What percentage of the human genome does the HLTH gene represent?
0.00005%.
What does 'Run to Red' refer to in agarose gel electrophoresis?
Setting up the apparatus.
What regions of the human genome are analyzed for DNA profiling?
Short tandem repeats (STR).
What should be done if a pre-stain was not applied?
Stain the gel.
How did the invention of PCR contribute to genetic research and forensics?
It enabled rapid advancements in both fields.
Who invented PCR and in what year?
Kary Mullis in 1985.
What role does the 3’-OH of the growing DNA strand play in DNA synthesis?
It attacks the α-PO4 of the next dNTP to be incorporated.
What is the role of the template DNA in each PCR reaction?
It is the DNA swabbed from individual poultry samples.
What is released during the formation of a phosphodiester bond?
Pyrophosphate (PPi).
How does PCR differ from DNA replication?
PCR aims to amplify a specific DNA sequence, not to copy the entire genome.
What is required to catalyze the addition of new nucleotides in PCR?
DNA polymerase (DNAP).
What is one application of PCR that requires high fidelity?
Cloning and other forms of genetic engineering.
What is a PCR application used for analyzing genetic mutations?
Mutational analysis.
Can you name a specific application for PCR outside of a research lab?
Forensic analysis, medical diagnostics, or paternity testing.
What is another application of PCR outside of a research lab?
Forensic analysis in criminal investigations.
What is the main purpose of PCR?
To amplify specific DNA sequences.
What is the function of helicase in DNA replication?
Unzips the double-stranded DNA.
What is done after pipetting the DNA/RNA sample into the wells?
Apply electricity and wait.
What are some stains used in agarose gel electrophoresis imaging?
Ethidium bromide and SYBRGreen.
What is done after pipetting the DNA/RNA sample into the wells?
Apply electricity and wait.
How many locations in the human genome are typically amplified and analyzed for DNA profiling?
20 locations.
What type of DNA sequence do the primers amplify in this PCR experiment?
A sequence that is unique to Salmonella species.
What equipment is used to visualize and image gels?
A UV transilluminator.
What role do the separated strands play in DNA synthesis?
Each strand acts as a template for synthesizing a new strand.
What type of linkage is formed during DNA synthesis?
A phosphodiester linkage between the new nucleotide and the growing DNA strand.
What is the result of the DNA synthesis process?
Two identical double-stranded DNA molecules, each with one original strand and one newly synthesized strand.
What does DNAP use to add the first dNTP during DNA synthesis?
The 3’-OH of the RNA primer.
What is another application of PCR that does not require high fidelity?
Analyzing whether a specific gene of interest is present in a sample.
How many errors does Taq polymerase introduce per every 44,000 bases added?
Approximately 1 error.
What are various additives used for in PCR?
To enhance PCR under difficult conditions, such as when the DNA template has a high GC content.
What is the process of DNA synthesis in living organisms called?
In vivo DNA synthesis.
What is the primary function of DNA polymerase in PCR?
To extend the DNA strands by adding free dNTPs.
What is agarose made of?
Repeating disaccharide units.
Why do nucleic acids move toward the anode in agarose gel electrophoresis?
Because they carry a strong negative charge.
In what applications are high-fidelity polymerases particularly important?
In applications requiring precise DNA replication.
How did PCR revolutionize forensic DNA analysis?
By allowing the amplification of small DNA samples for analysis.
What is the first step in the agarose gel electrophoresis process?
Cast the gel (Optional – apply a pre-stain).
What does 'in vivo' mean?
In living cells/organisms.
What do the primers in the PCR experiment amplify?
A sequence of DNA that is unique to Salmonella species.
What happens when UV light is applied to the stained gel?
The stain emits visible light, revealing the location of DNA/RNA.
What is a good negative control for the PCR experiment?
A 'mock' PCR reaction that does not contain any template DNA.
What other sources can DNA be extracted from for forensic analysis?
Hairs and skin cells left behind on objects ('touch' DNA).
What are the lanes in the agarose gel used for in this PCR experiment?
Lanes are used to show the DNA ladder, positive control, poultry samples, and negative control.
What is the length of the amplified fragment?
Slightly less than 150 bp (actual length is 137 bp).
Why is primase essential for DNA replication?
Because DNAP can only add on to an existing nucleic acid molecule.
What is meant by 'in vitro'?
Using biological molecules outside of their biological context.
What is a significant problem with Taq polymerase?
It has a high error rate.
If starting with a single molecule of template DNA, how many bases will Taq polymerase synthesize after 30 cycles for a 3000 bp amplicon?
More than 32 billion bp of DNA.
What type of primer is used in PCR?
Short DNA oligomers.
Which enzyme is responsible for unwinding the DNA during replication?
Helicase.
What can agarose gel electrophoresis be used to analyze?
PCR results, confirm integrity of nucleic acid samples, and generate genetic profiles.
What type of primer is used in PCR?
DNA primer.
What should you always run concurrently when setting up your gel?
A ladder of DNA standards.
What does the ladder of DNA standards include?
~10-15 different sized fragments of DNA.
Why do we compare our samples to the DNA ladder?
To determine the size of our fragment(s) and estimate the concentration based on band brightness.
From what bodily fluids can DNA be analyzed using PCR?
Blood, saliva, or semen.
What does the positive control in the PCR experiment indicate?
That the PCR reaction worked correctly and amplified the target DNA.
Which samples appear to be free from Salmonella?
Samples 9, 11, and 12.
What is the role of chemicals and reagents in PCR reactions?
They facilitate the amplification of DNA by providing necessary components for the reaction.
What is the length of the fragment amplified for Salmonella detection?
This would be determined by comparing the band size to the DNA ladder.
What chemical attributes of DNA affect its migration in agarose gel electrophoresis?
The size and charge of the DNA molecules influence their movement through the gel.
What role do primers play in PCR?
They initiate the extension process.
What is used as a template in PCR?
An existing DNA molecule.
What is the purpose of introducing bounds on DNA synthesis in PCR?
To copy a small portion of the DNA, not the entire molecule.
What serves as the template for DNA synthesis?
Cellular DNA.
What is the primary purpose of agarose gel electrophoresis?
To separate DNA or RNA molecules by size.
How do the stains used in agarose gel electrophoresis interact with nucleic acids?
The molecules intercalate into (interact with) nucleic acids.
What property do the staining molecules have?
Fluorescent properties.
Which lanes in the agarose gel indicate the presence of Salmonella?
Samples 3-5, 7, 8, and 10.
What happens to the two strands of a DNA molecule during synthesis?
They are separated (denatured).
What does it mean if you have unexpected bands in your gel analysis?
It could indicate contamination or non-specific amplification.
How can you determine which poultry samples contained Salmonella based on agarose gel results?
By comparing the bands in the lanes of the poultry samples to the positive control.
How is DNA evidence used in the justice system?
To identify criminals and exonerate wrongfully convicted individuals.
What does PCR stand for?
Polymerase Chain Reaction.
What is one application of PCR that does not require high fidelity?
Screening environmental samples or food products for the presence of E. coli or other pathogens.
What is the purpose of the buffer in the PCR reaction mix?
To maintain pH.
How do ions like Mg2+ assist in the PCR process?
They help primers anneal to the DNA template.
Does the error rate of Taq polymerase matter?
It depends on how you intend to use the amplified DNA.
What is required to initiate DNA synthesis?
An RNA primer (primase).
What initiates DNA denaturation in PCR?
Heat (thermal cycling).
What determines the pore size in agarose gel?
The concentration of agarose used (higher concentration = smaller pores).
How do molecules migrate through a gel matrix in agarose gel electrophoresis?
Molecules move through the gel based on their size and charge.
What are dNTPs and why are they included in the PCR reaction mix?
dNTPs are deoxynucleotide triphosphates, included in excess to provide building blocks for DNA synthesis.
What serves as the template in PCR?
Any DNA sample you want.
What is the method of DNA denaturation in PCR?
High heat (95°C).
What is a forensic application of PCR?
Forensics.
What is the function of the RNA primer in DNA synthesis?
It serves as the 'starting point' for DNA extension.
What is a specific application of PCR outside of a research lab?
Medical diagnostics, such as detecting viral infections.
