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MOLBIO LAB
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MOLBIO LAB
Quantification and Analysis of Proteins
Study
1
Question
What are the three major types of spectrophotometric assays for protein quantitation?
Answer
UV Absorbance Method, Colorimetric Dye-Binding Assay Method, UV Spectroscopy Method with A280
2
Question
What is the principle behind UV Spectroscopy Method with A280 for protein quantitation?
Answer
It detects amino acid residues with aromatic rings in a protein that absorb light, following the Beer-Lambert Law. It is used to detect proteins in the 20-3000 ug range.
3
Question
What are the advantages of UV Spectroscopy Method with A280 for protein quantitation?
Answer
Proteins are not lost in the process, not denatured, fast and easily automated, and it follows the sensitive Beer-Lambert Law.
4
Question
What are the drawbacks of UV Spectroscopy Method with A280 for protein quantitation?
Answer
Buffers and reagents can interfere with the spectrophotometric measurement at 280 nm.
5
Question
What is the principle behind UV Spectroscopy Method with A205 for protein quantitation?
Answer
It detects absorbance based on the peptide bonds in proteins, following the Beer-Lambert Law. It is used to detect proteins in the 1-100 ug/mL range.
6
Question
What are the advantages of UV Spectroscopy Method with A205 for protein quantitation?
Answer
It is around 7 times more sensitive than A280, proteins are not lost or denatured, and it is fast and easily automated.
7
Question
What are the colorimetric protein assay techniques mentioned in the text?
Answer
Biuret Reaction, Lowry Method, Bradford Method, Bicinchoninic Acid Assay (BCA) Method
8
Question
What is the principle of the Biuret Reaction in protein quantitation?
Answer
It involves complex formation of cupric ions with proteins in an alkaline solution, resulting in a purple-violet color. The amount of Cu-bathocuproine complex formed is inversely proportional to the protein concentration.
9
Question
How does the Lowry Method work for protein quantitation?
Answer
It involves adding phosphomolybdic phosphotungstic acid and Folin-Ciocalteu reagent to produce a blue-green color, with detection between 650-750 nm. It is based on the initial steps of the Biuret reaction.
10
Question
What is the principle of the Bradford Method for protein quantitation?
Answer
It involves the complex formation of Coomassie brilliant blue G250 dye with proteins, leading to a color change that can be quantitatively measured.
11
Question
What color is produced when the phenolic group of amino acid residues reduces phosphomolybdic acid?
Answer
Blue color
12
Question
What is the downside of the Bradford method in protein determination?
Answer
Staining cuvettes and forming precipitates that can interfere with readings; Destructive to proteins; Once the protein sample has reacted with the dye, it cannot be used for other assays
13
Question
What is the principle of the Bicinchoninic Acid Assay (BCA Method)?
Answer
Based on the initial steps of the Biuret reaction, adds bicinchoninic acid to produce a deep blue color under alkaline conditions
14
Question
What are the advantages of the Bicinchoninic Acid Assay (BCA Method)?
Answer
Compatible with detergents, less protein variability
15
Question
What are the drawbacks of the Bicinchoninic Acid Assay (BCA Method)?
Answer
Destructive to proteins, can be interfered by reducing and chelating agents like EDTA
16
Question
What is the principle of fluorescent dye-based assays in protein quantitation?
Answer
Amine labeling/derivatization using fluorescent probes to quantitate proteins containing lysine or free N-terminus
17
Question
What are the types of dyes used in fluorescent dye-based assays for protein quantitation?
Answer
Ophthalaldehyde (OPA), Fluorescamine, 3,4-carboxybenzyl quinoline-2-carboxyaldehyde (CBQCA)
18
Question
What are the different types of detection methods used in fluorescent dye-based assays?
Answer
Microplate and Cuvette
19
Question
What is the preferred dye for fluorescent detection in protein quantitation and why?
Answer
Ophthalaldehyde (OPA) reacts with thiol or cyanide, has greater solubility and stability
20
Question
What is the principle of column chromatography in protein separation?
Answer
Separation of proteins based on specific characteristics using a resin-filled column to separate proteins based on physical properties
21
Question
What are the different methods of column chromatography?
Answer
Size Exclusion Chromatography, Ion-Exchange Chromatography, Affinity Chromatography, and Hydrophobic Interaction Chromatography
22
Question
What is the principle of Size Exclusion Chromatography?
Answer
Separates proteins based on size and shape on the gel filtration column, smaller molecules travel slower through the pores, larger molecules travel faster
23
Question
How does Ion-Exchange Chromatography separate proteins?
Answer
According to their surface charges using resins with a particular charge to trap oppositely charged molecules, same charge molecules travel faster, opposite charge molecules travel slower
24
Question
What is the principle of Affinity Chromatography in protein separation?
Answer
Separates proteins according to their specific affinity for certain molecules or ligands on the column resin
25
Question
What is the principle behind Ion Exchange Chromatography?
Answer
Separates proteins according to their surface charges using resins with a particular charge to trap oppositely charged molecules.
26
Question
How do molecules with the same charge as the resin travel in Ion Exchange Chromatography?
Answer
They travel faster.
27
Question
What is the purpose of Anion-exchange resins in chromatography?
Answer
They attract negatively charged solutes.
28
Question
How does Affinity Chromatography separate proteins?
Answer
Based on specific affinity, where proteins that bind well to a ligand will attach while others pass through.
29
Question
What is the purpose of Hydrophobic Interaction Chromatography?
Answer
Separates proteins based on their hydrophobic groups using a Salting Out Method.
30
Question
What is the principle behind Electrophoresis?
Answer
Movement of molecules by electric current based on their charge.
Techniques in Sequencing
Study
1
Question
What is the purpose of sequencing DNA in molecular biology diagnostics?
Answer
To detect mutations, type microorganisms, determine identity and strains, identify human haplotypes, designate polymorphisms, and for DNA fingerprinting in forensics, among others.
2
Question
What is gene expression in molecular biology?
Answer
Gene expression refers to the process by which genetic information in DNA is used to encode a particular protein.
3
Question
What are the techniques in sequencing mentioned in the text?
Answer
Manual Sequencing, Maxam Gilbert Method, Dideoxy Chain Termination, Automated DNA Sequencing, Next-Generation Sequencing, Pyrosequencing, Roche 454, Illumina, Solexa, Ion Torrent, ABi SOLiD, Third-Generation Sequencing, Single-Molecule Sequencing, Helicos, PacBio, Nanopore Sequencing, Oxford Nanopore.
4
Question
What is the Maxam-Gilbert method of sequencing based on?
Answer
The Maxam-Gilbert method of sequencing is based on chemical modification of DNA and subsequent cleavage at specific bases.
5
Question
How does manual sequencing using Maxam-Gilbert method work?
Answer
The sample is radioactively labeled, divided into four tubes, treated with base modifiers, and fragments are separated by size in polyacrylamide gel electrophoresis.
6
Question
What are the disadvantages of manual sequencing using the Maxam-Gilbert method?
Answer
It cannot read large sequences, only fragments of DNA; it utilizes a radioactively labeled sample that can pose health risks for researchers.
7
Question
What is chain termination sequencing also known as?
Answer
Chain termination sequencing is also known as dideoxy sequencing or Sanger sequencing.
8
Question
Who revolutionized the concept of sequencing by first determining the amino acid sequence of insulin?
Answer
Frederick Sanger revolutionized the concept of sequencing by determining the amino acid sequence of insulin.
9
Question
What are the components of chain termination sequencing?
Answer
Single-stranded DNA template sample, sequencing primer, DNA polymerase, radioactively labeled nucleotides (dNTPs), chain terminating nucleotides (ddNTPs).
10
Question
What is the principle behind chain termination sequencing?
Answer
DNA polymerase will build DNA chains one by one, but in each sequence it will stop after incorporating a chain terminating nucleotide.
11
Question
What are the components involved in chain termination sequencing?
Answer
Single-stranded DNA template sample, Sequencing primer, DNA polymerase, Radioactive-labeled nucleotides (dNTPs), Chain-terminating nucleotides (ddNTPs)
12
Question
What is the procedure for chain termination sequencing?
Answer
1. Divide the template into four tubes with different chain-terminating nucleotides. 2. Anneal the sequencing primer. 3. Initiate DNA synthesis through DNA polymerase.
13
Question
What are the pros and cons of using the Klenow fragment of DNA polymerase in sequencing?
Answer
Pros: No exonuclease activity. Cons: Not thermostable.
14
Question
How are DNA fragments separated in chain termination sequencing using PAGE?
Answer
DNA fragments are separated by size using Polyacrylamide Gel Electrophoresis (PAGE), with the bottom band representing the first sequence and the band near the wells representing the last sequence.
15
Question
What is automated DNA sequencing, and how does it differ from manual sequencing?
Answer
Automated DNA sequencing uses fluorescence labels and capillary electrophoresis instead of radioactive labels and PAGE. It also employs computer software for recording reads, making the process less laborious and error-prone compared to manual sequencing.
16
Question
What is Next Generation Sequencing (NGS) and what major advantage does it offer over manual sequencing?
Answer
NGS is a high-throughput sequencing method that allows for automated massively parallel sequencing, enabling millions of sequencing reactions to occur and be recorded simultaneously, unlike manual sequencing.
17
Question
What is pyrosequencing, and how does it detect nucleotide incorporation?
Answer
Pyrosequencing detects pyrophosphate released during DNA synthesis. For every nucleotide incorporated, a pyrophosphate is released and detected by ATP sulfurylase and luciferase, producing light that is recorded by the machine.
18
Question
What is the principle behind Illumina Solexa sequencing, and how does it differ from traditional sequencing methods?
Answer
Illumina Solexa sequencing uses reversible dye terminators, where synthesis stops upon attachment to the growing DNA sequence and fluoresces. Unlike traditional methods, all DNA fragments in Illumina Solexa sequencing are of equal length.
19
Question
What is the principle used in ILLUMINA SOLEXA sequencing?
Answer
Reversible DyeTermination
20
Question
How does the reversible terminator work in ILLUMINA SOLEXA sequencing?
Answer
It attaches to the growing DNA sequence, causing synthesis to stop and fluoresce, then leaves to allow synthesis to continue.
21
Question
What is the advantage of ION TORRENT sequencing?
Answer
It can read many sequences.
22
Question
What is the disadvantage of ION TORRENT sequencing?
Answer
It lacks sensitivity due to pH being affected by multiple factors.
23
Question
What is the principle used in ABi SOLiD sequencing?
Answer
Sequencing by Ligation
24
Question
How does ABi SOLiD sequencing detect nucleotides in the growing sequence?
Answer
It ligates two oligonucleotides in the growing sequence, which fluoresce depending on the color of the oligonucleotide.
25
Question
What are the four colors used in ABi SOLiD sequencing to indicate different bases?
Answer
Blue, green, yellow, red
26
Question
What is a key characteristic of Third Generation Sequencing?
Answer
PCR is not needed before sequencing.
27
Question
How is signal captured in real time in Third Generation Sequencing?
Answer
Signal is captured in real time during sequencing.
28
Question
What is the main characteristic of Single Molecule Sequencing?
Answer
Each nucleotide corresponds to a particular probe, and signal is captured for each nucleotide in real time.
29
Question
What is the key concept of Nanopore sequencing?
Answer
Utilization of nanopore to detect ionic flow and current disruptions, which are interpreted as sequence data.
30
Question
What is a key feature of Second Generation sequencing?
Answer
Short range sequencing with higher sensitivity and specificity.
Amplification of Nucleic Acids
Study
1
Question
What is PCR short for?
Answer
Polymerase Chain Reaction
2
Question
What is the process of PCR cycles of temperature changes at fixed time intervals?
Answer
Thermocycling
3
Question
What are the 3 distinct temperatures involved in PCR?
Answer
Denaturation, Annealing, Extension (Elongation)
4
Question
What is used in PCR to create numerous copies of DNA from DNA building blocks called Deoxynucleoside triphosphates?
Answer
Thermostable DNA polymerase
5
Question
What is the PCR machine called that is used for amplifying DNA fragments?
Answer
Thermocycler
6
Question
What is the amplified product in PCR known as?
Answer
Amplicon
7
Question
What is the purpose of genetic testing using PCR?
Answer
To determine parentage or lineage of a child
8
Question
What can PCR be used for in pathogen detection?
Answer
Detecting the presence of pathogens like TB or SARS-CoV2 RNA
9
Question
How is PCR used in drug development?
Answer
To create new drugs, like against TB or salmonella
10
Question
What is the application of PCR in crop modification?
Answer
Creating genetically modified organisms (GMOs)
11
Question
In forensic analysis, how is PCR utilized?
Answer
To analyze DNA evidence for legal cases
12
Question
What can be determined through PCR sequencing?
Answer
The origin of infections, like influenza, from sources like pigs, bats, or avians
13
Question
What are the key reagents used in PCR experiments?
Answer
Template DNA, Oligonucleotide Primers, DNA Polymerase, Deoxynucleotide Triphosphates (dNTPS), Buffer with MgCl2
14
Question
What is the purpose of template DNA in PCR?
Answer
To serve as the sample from which the DNA fragment will be amplified
15
Question
What are the sense and antisense strands of DNA template also known as?
Answer
Coding strand (sense) and complementary strand (antisense)
16
Question
What is the function of oligonucleotide primers in PCR?
Answer
To prime the polymerase reaction by binding to specific DNA sequences
17
Question
What is the function of a Forward/Sense Primer in polymerase chain reaction?
Answer
A primer that binds to the antisense strand and has the same sequence as the sense strand.
18
Question
What is the function of a Reverse/Antisense Primer in polymerase chain reaction?
Answer
A primer that binds to the sense strand and has a sequence that is reversed and complementary to the sense strand.
19
Question
What are the considerations in choosing primers for PCR?
Answer
1. Primers must be complementary to the 5' and 3' regions of the DNA template. 2. Primers should be between 15 and 30 base pairs long with about 50% guanine and cytosine content. 3. The melting temperatures of both primers should be above 50-60°C. 4. Primers should not be complementary to each other to avoid primer dimers. 5. Primers should not contain secondary structures.
20
Question
What is the role of DNA polymerase in PCR?
Answer
DNA polymerase are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA.
21
Question
Which DNA polymerase is commonly used in PCR and why?
Answer
Taq polymerase, isolated from the bacterium Thermus aquaticus. It is thermostable and can withstand high temperatures, making it suitable for PCR.
22
Question
What is the disadvantage of Taq polymerase in terms of error rate?
Answer
Taq polymerase has an error rate of 1 per 3300 bases compared to other polymerases with higher fidelity.
23
Question
What is meant by high fidelity in DNA polymerase?
Answer
High fidelity means that the DNA bases are correctly paired to one another, such as Adenine with Thymine and Guanine with Cytosine.
24
Question
What are dNTPs and their role in PCR?
Answer
dNTPs are deoxynucleotide triphosphates, the building blocks of DNA that comprise the base pairs in the growing DNA strands during PCR.
25
Question
What is the purpose of the buffer containing MgCl2 in PCR?
Answer
The buffer maintains pH, stabilizes the reaction, provides important ions, and cofactors to the polymerase enzyme during PCR.
26
Question
Why is PCR grade water used in PCR reactions?
Answer
PCR grade water is free of ions that can inhibit the reaction and is used as a solvent in PCR reactions.
27
Question
What does a Colored master mix contain?
Answer
28
Question
Answer
Hot start Taq polymerase, dNTPs, MgCl2, and KCl
29
Question
What is the purpose of a master mix in PCR reactions?
Answer
To scale up the reaction by creating a large volume mixture of all reagents shared among samples
30
Question
What is the first step of a PCR reaction?
Answer
Denaturation, heating the mixture to 94°C to break hydrogen bonds and denature the DNA into single-stranded molecules