p.16
Applications of PCR in Forensics
What is one key benefit of using PCR in forensic science?
It enables the analysis of degraded or limited DNA samples.
p.5
Biochemical Principles of DNA Synthesis
What does primase do during DNA synthesis?
Generates an RNA primer from which DNAP can initiate the extension process.
p.2
Invention and Impact of PCR
What is the challenge faced in studying the HLTH gene?
The gene must be present in high abundance for mutational analysis.
p.9
Invention and Impact of PCR
What does PCR stand for?
Polymerase Chain Reaction.
p.27
Applications of PCR in Forensics
What is the purpose of using PCR in this experiment?
To determine if there is Salmonella contamination in poultry samples.
p.25
Applications of PCR in Forensics
What is the purpose of using PCR in the context of poultry samples?
To determine if there is Salmonella contamination.
p.3
Invention and Impact of PCR
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.
p.6
Biochemical Principles of DNA Synthesis
What is required for DNA synthesis in vivo?
Free deoxynucleoside triphosphates (dNTPs) for each of the 4 bases.
p.26
Analyzing PCR Results with Gel Electrophoresis
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.
p.18
DNA Profiling and Criminal Investigations
What database can DNA profiles be compared to?
The FBI’s CODIS (Combined DNA Index System).
p.24
Principles of Agarose Gel Electrophoresis
What is the significance of using a DNA ladder in the gel electrophoresis?
To determine the size of the amplified DNA fragments.
p.16
Invention and Impact of PCR
What does PCR stand for?
Polymerase Chain Reaction.
p.16
DNA Profiling and Criminal Investigations
Why is PCR important for matching DNA in criminal investigations?
It increases the quantity of DNA available for comparison.
p.17
Applications of PCR in Forensics
What were the limitations of forensic analysis before the invention of PCR?
Limited to fingerprinting, blood typing, and obvious physical evidence.
p.24
PCR Reaction Mix Components
What is the purpose of designing primers in a PCR experiment for Salmonella detection?
To amplify a DNA sequence unique to Salmonella species.
p.24
PCR Reaction Mix Components
What component of the PCR setup needs to be changed for each sample tested?
The DNA template from the poultry samples.
p.24
PCR Reaction Mix Components
What would be a good positive control for the PCR experiment?
A sample known to contain Salmonella DNA.
p.1
Biochemical Principles of DNA Synthesis
What are the biochemical principles underlying DNA synthesis?
They involve the mechanisms and processes that facilitate the formation of DNA molecules.
p.3
In Vivo vs. In Vitro DNA Synthesis
What is the primary goal of PCR?
To isolate and amplify a small piece of DNA that we are interested in analyzing.
p.12
PCR Reaction Mix Components
What type of primers are included in the PCR reaction mix?
Forward and reverse primers (in excess).
p.11
Biochemical Principles of DNA Synthesis
What is the method of DNA denaturation in cellular replication?
Helicase unwinds the DNA.
p.7
Biochemical Principles of DNA Synthesis
What are the building blocks used by DNA polymerase during DNA synthesis?
Free dNTPs (deoxynucleotide triphosphates).
p.10
In Vivo vs. In Vitro DNA Synthesis
What serves as the template in PCR?
The DNA sample being amplified.
p.20
Principles of Agarose Gel Electrophoresis
What is the first step in the agarose gel electrophoresis process?
Cast the gel (Optional – apply a pre-stain).
p.17
Applications of PCR in Forensics
What was a major challenge in using DNA for solving crimes before PCR?
Analytical techniques required a larger sample than was usually possible.
p.17
Applications of PCR in Forensics
How does PCR assist in DNA analysis?
It allows us to multiply a tiny sample of DNA for analysis.
p.18
DNA Profiling and Criminal Investigations
What does the analysis of STRs reveal?
The length of these STRs for that person.
p.27
Analyzing PCR Results with Gel Electrophoresis
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.
p.3
Biochemical Principles of DNA Synthesis
What is DNA amplification?
The process that dramatically increases the copy number of a specific sequence of DNA in a sample.
p.4
Biochemical Principles of DNA Synthesis
How are complementary nucleotides added during DNA synthesis?
In sequence to the nascent DNA strand (A—T; C—G).
p.1
Principles of Agarose Gel Electrophoresis
What is agarose gel electrophoresis used for?
It is used to separate DNA molecules based on size.
p.12
PCR Reaction Mix Components
What ions are typically added to the PCR reaction mix, and why?
MgCl2 is usually added; Mg2+ is an important cofactor for DNA polymerase.
p.19
Principles of Agarose Gel Electrophoresis
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.
p.5
In Vivo vs. In Vitro DNA Synthesis
What does 'in vivo' mean?
In living cells/organisms.
p.2
Invention and Impact of PCR
What is the primary need for PCR in biomedical research?
To isolate and copy specific segments of DNA, such as a gene of interest.
p.20
Principles of Agarose Gel Electrophoresis
How is stained DNA visualized in agarose gel electrophoresis?
Using UV light to illuminate the stained DNA.
p.2
Invention and Impact of PCR
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.
p.26
PCR Reaction Mix Components
What is a good positive control for the PCR experiment?
A known sample of Salmonella DNA as a template.
p.26
Analyzing PCR Results with Gel Electrophoresis
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.
p.24
PCR Reaction Mix Components
What would be a good negative control for the PCR experiment?
A sample without any DNA or a sample known not to contain Salmonella.
p.1
In Vivo vs. In Vitro DNA Synthesis
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.
p.22
Principles of Agarose Gel Electrophoresis
How does the gel documentation system record an image of the gel?
It uses a camera with UV filters.
p.25
Principles of Agarose Gel Electrophoresis
What is the significance of the DNA ladder in the gel image?
It serves as a size reference for the PCR products.
p.11
In Vivo vs. In Vitro DNA Synthesis
What is the primary function of DNA polymerase in cellular replication?
To extend DNA strands during replication.
p.15
Applications of PCR in Forensics
What is a quality control application of PCR?
Quality control analysis in consumer products and medications.
p.15
High-Fidelity Polymerases and Their Importance
How is PCR used in paternity testing?
To analyze genetic markers for parentage.
p.19
Principles of Agarose Gel Electrophoresis
How does agarose gel form?
It polymerizes into a solid gel when dissolved in an aqueous solution.
p.14
High-Fidelity Polymerases and Their Importance
What is the primary purpose of high-fidelity polymerases?
To create an exact, error-free copy of DNA.
p.5
Biochemical Principles of DNA Synthesis
What is the role of DNA polymerase (DNAP) in DNA synthesis?
Adds complementary nucleotides to the growing DNA strand (extension).
p.20
Principles of Agarose Gel Electrophoresis
What does 'Run to Red' refer to in agarose gel electrophoresis?
It refers to setting up the apparatus correctly.
p.16
DNA Profiling and Criminal Investigations
What role does PCR play in identifying suspects?
It helps in generating DNA profiles from evidence found at crime scenes.
p.26
Applications of PCR in Forensics
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.
p.4
Biochemical Principles of DNA Synthesis
In which direction is DNA synthesized?
In the 5’ to 3’ direction.
p.4
Biochemical Principles of DNA Synthesis
What type of process is DNA synthesis?
A semi-conservative process.
p.23
Analyzing PCR Results with Gel Electrophoresis
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.
p.18
DNA Profiling and Criminal Investigations
What is unique to every individual in DNA profiling?
The DNA profile created from STR analysis.
p.23
Analyzing PCR Results with Gel Electrophoresis
What should you consider if you do not have a DNA fragment at all?
Your PCR may have failed.
p.25
Analyzing PCR Results with Gel Electrophoresis
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.
p.11
High-Fidelity Polymerases and Their Importance
What type of DNA polymerase is used in PCR?
Thermostable DNA polymerase (Taq polymerase).
p.8
Biochemical Principles of DNA Synthesis
What acts as the substrate for DNAP in PCR?
Free deoxynucleotide triphosphates (dNTPs).
p.8
In Vivo vs. In Vitro DNA Synthesis
How does PCR differ from natural DNA replication?
PCR uses only DNAP and introduces bounds on DNA synthesis.
p.11
In Vivo vs. In Vitro DNA Synthesis
What is the function of primers in both PCR and cellular replication?
To serve as the starting point of DNA extension.
p.10
In Vivo vs. In Vitro DNA Synthesis
What is the primary function of DNA polymerase in cellular replication?
To synthesize new DNA strands by adding nucleotides.
p.19
Principles of Agarose Gel Electrophoresis
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.
p.21
Principles of Agarose Gel Electrophoresis
What does 'Run to Red' refer to in agarose gel electrophoresis?
Setting up the apparatus.
p.18
DNA Profiling and Criminal Investigations
What regions of the human genome are analyzed for DNA profiling?
Short tandem repeats (STR).
p.1
Invention and Impact of PCR
How did the invention of PCR contribute to genetic research and forensics?
It enabled rapid advancements in both fields.
p.6
Biochemical Principles of DNA Synthesis
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.
p.25
PCR Reaction Mix Components
What is the role of the template DNA in each PCR reaction?
It is the DNA swabbed from individual poultry samples.
p.3
In Vivo vs. In Vitro DNA Synthesis
How does PCR differ from DNA replication?
PCR aims to amplify a specific DNA sequence, not to copy the entire genome.
p.15
High-Fidelity Polymerases and Their Importance
What is one application of PCR that requires high fidelity?
Cloning and other forms of genetic engineering.
p.11
Applications of PCR in Forensics
Can you name a specific application for PCR outside of a research lab?
Forensic analysis, medical diagnostics, or paternity testing.
p.10
Applications of PCR in Forensics
What is another application of PCR outside of a research lab?
Forensic analysis in criminal investigations.
p.9
Invention and Impact of PCR
What is the main purpose of PCR?
To amplify specific DNA sequences.
p.5
Biochemical Principles of DNA Synthesis
What is the function of helicase in DNA replication?
Unzips the double-stranded DNA.
p.20
Principles of Agarose Gel Electrophoresis
What is done after pipetting the DNA/RNA sample into the wells?
Apply electricity and wait.
p.22
Principles of Agarose Gel Electrophoresis
What are some stains used in agarose gel electrophoresis imaging?
Ethidium bromide and SYBRGreen.
p.21
Principles of Agarose Gel Electrophoresis
What is done after pipetting the DNA/RNA sample into the wells?
Apply electricity and wait.
p.25
PCR Reaction Mix Components
What type of DNA sequence do the primers amplify in this PCR experiment?
A sequence that is unique to Salmonella species.
p.4
In Vivo vs. In Vitro DNA Synthesis
What role do the separated strands play in DNA synthesis?
Each strand acts as a template for synthesizing a new strand.
p.6
Biochemical Principles of DNA Synthesis
What type of linkage is formed during DNA synthesis?
A phosphodiester linkage between the new nucleotide and the growing DNA strand.
p.4
In Vivo vs. In Vitro DNA Synthesis
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.
p.6
In Vivo vs. In Vitro DNA Synthesis
What does DNAP use to add the first dNTP during DNA synthesis?
The 3’-OH of the RNA primer.
p.15
Applications of PCR in Forensics
What is another application of PCR that does not require high fidelity?
Analyzing whether a specific gene of interest is present in a sample.
p.12
PCR Reaction Mix Components
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.
p.10
In Vivo vs. In Vitro DNA Synthesis
What is the primary function of DNA polymerase in PCR?
To extend the DNA strands by adding free dNTPs.
p.19
Principles of Agarose Gel Electrophoresis
What is agarose made of?
Repeating disaccharide units.
p.19
Principles of Agarose Gel Electrophoresis
Why do nucleic acids move toward the anode in agarose gel electrophoresis?
Because they carry a strong negative charge.
p.14
High-Fidelity Polymerases and Their Importance
In what applications are high-fidelity polymerases particularly important?
In applications requiring precise DNA replication.
p.16
Applications of PCR in Forensics
How did PCR revolutionize forensic DNA analysis?
By allowing the amplification of small DNA samples for analysis.
p.21
Principles of Agarose Gel Electrophoresis
What is the first step in the agarose gel electrophoresis process?
Cast the gel (Optional – apply a pre-stain).
p.4
In Vivo vs. In Vitro DNA Synthesis
What does 'in vivo' mean?
In living cells/organisms.
p.27
PCR Reaction Mix Components
What do the primers in the PCR experiment amplify?
A sequence of DNA that is unique to Salmonella species.
p.22
Principles of Agarose Gel Electrophoresis
What happens when UV light is applied to the stained gel?
The stain emits visible light, revealing the location of DNA/RNA.
p.26
PCR Reaction Mix Components
What is a good negative control for the PCR experiment?
A 'mock' PCR reaction that does not contain any template DNA.
p.17
Applications of PCR in Forensics
What other sources can DNA be extracted from for forensic analysis?
Hairs and skin cells left behind on objects ('touch' DNA).
p.26
Principles of Agarose Gel Electrophoresis
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.
p.27
Biochemical Principles of DNA Synthesis
What is the length of the amplified fragment?
Slightly less than 150 bp (actual length is 137 bp).
p.6
In Vivo vs. In Vitro DNA Synthesis
Why is primase essential for DNA replication?
Because DNAP can only add on to an existing nucleic acid molecule.
p.8
In Vivo vs. In Vitro DNA Synthesis
What is meant by 'in vitro'?
Using biological molecules outside of their biological context.
p.13
High-Fidelity Polymerases and Their Importance
What is a significant problem with Taq polymerase?
It has a high error rate.
p.13
High-Fidelity Polymerases and Their Importance
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.
p.19
Principles of Agarose Gel Electrophoresis
What can agarose gel electrophoresis be used to analyze?
PCR results, confirm integrity of nucleic acid samples, and generate genetic profiles.
p.23
Principles of Agarose Gel Electrophoresis
What should you always run concurrently when setting up your gel?
A ladder of DNA standards.
p.23
Principles of Agarose Gel Electrophoresis
What does the ladder of DNA standards include?
~10-15 different sized fragments of DNA.
p.23
Analyzing PCR Results with Gel Electrophoresis
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.
p.25
Analyzing PCR Results with Gel Electrophoresis
What does the positive control in the PCR experiment indicate?
That the PCR reaction worked correctly and amplified the target DNA.
p.1
Components and Functions in PCR Reactions
What is the role of chemicals and reagents in PCR reactions?
They facilitate the amplification of DNA by providing necessary components for the reaction.
p.24
Analyzing PCR Results with Gel Electrophoresis
What is the length of the fragment amplified for Salmonella detection?
This would be determined by comparing the band size to the DNA ladder.
p.1
Principles of Agarose Gel Electrophoresis
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.
p.8
Biochemical Principles of DNA Synthesis
What role do primers play in PCR?
They initiate the extension process.
p.8
Biochemical Principles of DNA Synthesis
What is used as a template in PCR?
An existing DNA molecule.
p.8
In Vivo vs. In Vitro DNA Synthesis
What is the purpose of introducing bounds on DNA synthesis in PCR?
To copy a small portion of the DNA, not the entire molecule.
p.19
Principles of Agarose Gel Electrophoresis
What is the primary purpose of agarose gel electrophoresis?
To separate DNA or RNA molecules by size.
p.22
Principles of Agarose Gel Electrophoresis
How do the stains used in agarose gel electrophoresis interact with nucleic acids?
The molecules intercalate into (interact with) nucleic acids.
p.27
Analyzing PCR Results with Gel Electrophoresis
Which lanes in the agarose gel indicate the presence of Salmonella?
Samples 3-5, 7, 8, and 10.
p.4
In Vivo vs. In Vitro DNA Synthesis
What happens to the two strands of a DNA molecule during synthesis?
They are separated (denatured).
p.23
Analyzing PCR Results with Gel Electrophoresis
What does it mean if you have unexpected bands in your gel analysis?
It could indicate contamination or non-specific amplification.
p.24
Analyzing PCR Results with Gel Electrophoresis
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.
p.18
DNA Profiling and Criminal Investigations
How is DNA evidence used in the justice system?
To identify criminals and exonerate wrongfully convicted individuals.
p.8
In Vivo vs. In Vitro DNA Synthesis
What does PCR stand for?
Polymerase Chain Reaction.
p.15
Applications of PCR in Forensics
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.
p.12
PCR Reaction Mix Components
How do ions like Mg2+ assist in the PCR process?
They help primers anneal to the DNA template.
p.13
High-Fidelity Polymerases and Their Importance
Does the error rate of Taq polymerase matter?
It depends on how you intend to use the amplified DNA.
p.19
Principles of Agarose Gel Electrophoresis
What determines the pore size in agarose gel?
The concentration of agarose used (higher concentration = smaller pores).
p.1
Principles of Agarose Gel Electrophoresis
How do molecules migrate through a gel matrix in agarose gel electrophoresis?
Molecules move through the gel based on their size and charge.
p.12
PCR Reaction Mix Components
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.
p.7
Biochemical Principles of DNA Synthesis
What is the function of the RNA primer in DNA synthesis?
It serves as the 'starting point' for DNA extension.
p.10
Applications of PCR in Forensics
What is a specific application of PCR outside of a research lab?
Medical diagnostics, such as detecting viral infections.
