Protein Sequencing Techniques and PCR

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Questions and Answers

What is the maximum length of peptides that can be reliably sequenced using Edman degradation?

Peptides longer than about 50-70 amino acids cannot be sequenced reliably by Edman degradation.

Which enzymes are typically used to destabilize long protein chains for sequencing?

Endopeptidases such as trypsin or pepsin, and chemical reagents like cyanogen bromide are used.

Describe the role of mass spectrometry in protein sequencing.

Mass spectrometry sequences proteins by measuring the mass-charge ratios of peptide fragments.

How does Edman degradation automate the sequencing process?

Each cycle releases and derivatizes one amino acid from the peptide's N-terminus, which is then identified by HPLC. Signup and view all the answers

What is the primary advantage of using mass spectrometry in protein analysis compared to Edman degradation?

Mass spectrometry provides high throughput automation and greater precision in protein analysis. Signup and view all the answers

What is the purpose of comparing mass spectrometry results against a protein database?

The analysis helps determine the sequences of the peptide fragments based on previously sequenced proteins. Signup and view all the answers

What happens to peptide fragments in the mass spectrometer during mass spectrometry?

The peptides are fragmented until only single ions remain, which are then analyzed for mass-charge ratios. Signup and view all the answers

Why is Edman degradation still used alongside mass spectrometry in protein sequencing?

Edman degradation complements mass spectrometry in difficult protein identifications. Signup and view all the answers

What is the role of DNA polymerase in the extension phase of the PCR process?

DNA polymerase attaches to the primers and elongates the new DNA strands by adding dNTPs. Signup and view all the answers

At what temperature range does the elongation phase of PCR typically occur?

The elongation phase typically occurs at 60 – 75 °C. Signup and view all the answers

How many new DNA sequences are formed after one cycle of PCR starting with one original DNA molecule?

Two new DNA sequences are formed after one cycle of PCR. Signup and view all the answers

What type of results does conventional PCR provide, and how are these results typically analyzed?

Conventional PCR provides qualitative results, which are analyzed using agarose gel electrophoresis. Signup and view all the answers

What is the basic structure of a PCR cycle?

A PCR cycle consists of denaturation, annealing, and extension steps. Signup and view all the answers

What is the significance of using primers in the PCR process?

Primers are essential as they provide a starting point for DNA polymerase to begin DNA synthesis. Signup and view all the answers

What is the purpose of the denaturation step in the PCR cycle?

The denaturation step serves to separate the double-stranded DNA into single strands. Signup and view all the answers

What is the expected number of DNA copies after 30 cycles of PCR, starting with a single copy?

The expected number of DNA copies would be $2^{30}$ after 30 cycles. Signup and view all the answers

What is one key application of qPCR in the field of diagnostics?

Pathogen detection or viral quantification. Signup and view all the answers

Explain the difference between qPCR and RT-qPCR.

qPCR quantifies DNA, while RT-qPCR involves reverse transcription to quantify RNA. Signup and view all the answers

What does the Cycle Threshold (Ct) value indicate in qPCR?

Ct indicates the number of cycles required for a fluorescent signal to cross the threshold. Signup and view all the answers

Name one fluorescent dye used for sequence-unspecific detection in qPCR.

SYBR® Green I. Signup and view all the answers

What is gene expression profiling in the context of qPCR?

Gene expression profiling assesses the levels of mRNA to determine gene activity. Signup and view all the answers

How can qPCR be used for SNP genotyping?

qPCR can amplify specific alleles, allowing for discrimination between different SNPs. Signup and view all the answers

In what way can qPCR contribute to somatic mutation studies?

qPCR can detect and quantify mutations in specific genes involved in cancer. Signup and view all the answers

What role does chromatin IP quantification play in qPCR?

It quantifies specific DNA regions bound by proteins, indicating regulatory interactions. Signup and view all the answers

What is the formula for calculating amplification efficiency (E) in PCR?

Amplification efficiency (E) is calculated as $E = 10^{-1/slope}$. Signup and view all the answers

What percentage range is considered optimal for amplification efficiency in PCR?

The desired amplification efficiency range is from 90% to 110%. Signup and view all the answers

How is the ratio of target gene molecules calculated in a PCR cycle?

It is calculated by dividing the number of target gene molecules at the end of the PCR cycle by those at the start of the same cycle. Signup and view all the answers

What does a GAPDH Ct value represent in the context of quantifying TNF-alpha expression?

The GAPDH Ct value serves as a reference point for normalizing the TNF-alpha Ct value. Signup and view all the answers

Why is it important to determine the expression of TNF-alpha?

Determining TNF-alpha expression is important for understanding its role in inflammation and immune responses. Signup and view all the answers

What kind of contaminants and microorganisms were focused on in the study mentioned in the content?

The study focused on the carcinogen aflatoxin and its main fungal contaminant, Aspergillus section Flavi spp. Signup and view all the answers

What do Ct values indicate in a PCR experiment?

Ct values indicate the threshold cycle at which the fluorescent signal can first be detected, reflecting the amount of initial template. Signup and view all the answers

What is the significance of using nanobody-phage systems in the detection of contaminants?

Nanobody-phage systems enhance sensitivity and specificity in detecting contaminants like aflatoxin. Signup and view all the answers

How does the placement of the membrane affect the movement of proteins in electrophoretic transfer?

The membrane must be placed in the positive electrode, allowing negatively charged proteins to move from the gel to the membrane when voltage is applied. Signup and view all the answers

What is the purpose of blocking in the context of antibody binding to a membrane?

Blocking prevents antibodies from binding nonspecifically to the membrane surface. Signup and view all the answers

Describe the role of primary and secondary antibodies in protein detection.

Primary antibodies bind specifically to the target protein, while secondary antibodies are conjugated with enzymes to produce a detectable signal. Signup and view all the answers

What is ELISA and what types of molecules can it detect?

ELISA is an immunoassay used to detect and quantify small molecules like antigens, antibodies, peptides, or proteins. Signup and view all the answers

What are the main reagents used in an ELISA?

The main reagents are the antigen, antibody, enzyme-conjugated antibody, and substrate. Signup and view all the answers

How does the polymerase chain reaction (PCR) amplify DNA?

PCR amplifies a small amount of target DNA from a template into millions of copies through repeated thermal cycling. Signup and view all the answers

What is the significance of washing in the ELISA process?

Washing removes unbound antibodies from the assay, ensuring that only specific binding contributes to the signal. Signup and view all the answers

What are some applications of PCR technology?

PCR is used in diagnosis, therapeutic development, genotyping, sequencing, and forensic analysis. Signup and view all the answers

What are antisense oligonucleotides used for in molecular biology?

Antisense oligonucleotides are used to bind to complementary RNA sequences, inhibiting gene expression. Signup and view all the answers

Explain the function of small interfering RNA (siRNA).

siRNA is involved in RNA interference, where it targets and degrades specific mRNA molecules, preventing protein production. Signup and view all the answers

Describe the role of primers in DNA sequencing.

Primers are short nucleic acid sequences that provide a starting point for DNA synthesis during amplification and sequencing. Signup and view all the answers

What is the purpose of probes in detecting complementary DNA or RNA?

Probes hybridize to specific nucleic acid sequences, allowing for the detection and quantification of target DNA or RNA. Signup and view all the answers

How can oligonucleotides be used for targeted mutation introduction?

Oligonucleotides can be designed to introduce specific mutations at target locations in a DNA sequence via homologous recombination. Signup and view all the answers

What was the significance of the Merrifield Method in peptide synthesis?

The Merrifield Method revolutionized peptide synthesis by allowing for solid-phase synthesis, improving yield and efficiency. Signup and view all the answers

In what way do oligo therapeutics and gene therapy utilize oligonucleotides?

Oligo therapeutics and gene therapy employ oligonucleotides to modify gene expression or provide therapeutic effects by targeting specific sequences. Signup and view all the answers

What is the primary difference between liquid-phase and solid-phase peptide synthesis?

Liquid-phase synthesis is classical and suitable for large-scale production, while solid-phase synthesis allows for faster production with higher yields. Signup and view all the answers

Study Notes

### Electrophoresis 1

Electrophoresis is a technique used to separate and sometimes purify charged macromolecules that differ in size, charge and conformation in an electric field.
When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge.
The greater the charge of a macromolecule, the faster it migrates (greater electrophoretic mobility).
Greater size of the macromolecule will have greater frictional and electrostatic forces, slower electrophoretic mobility.
Rounded molecules have lesser frictional and electrostatic retardation compared to non-globular structure.
Globular proteins move faster than fibrous proteins; supercoiled plasmid move faster than linear plasmid.
DNA (and RNA) molecules are negatively charged, when placed in an electric field, they migrate to the positively charged anode.
DNA fragments (double-stranded linear DNA) have a uniform mass/charge ratio; thus, DNA fragments are separated by size within an agarose gel.
Shorter fragments migrate faster, longer fragments migrate slower.
The distance migrated on a gel correlates inversely with the logarithm of molecular weight.
Proteins and nucleic acids migrate within a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel.
These are solid yet porous matrixes.
Molecules will move through the matrix at different rates, usually determined by their mass, charge and conformation; they eventually get separated as different 'bands'.
Molecular weight markers are run side by side with samples; the molecular weight of samples can be estimated by referring to the markers.
DNA electrophoresis often uses agarose gel.
Protein electrophoresis often uses polyacrylamide gel.

Electrophoresis 2

SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a technique used to resolve and separate proteins based on molecular weight.
Proteins are mixed with Laemmli buffer before electrophoresis.
Laemmli buffer contains SDS (an anionic detergent), 2-mercaptoethanol (breaks disulfide bonds in globular proteins), and glycerol (high density solution to pull protein sample down and stay in its designated well).
SDS gives all proteins an overall negative charge proportional to its mass
2-mercaptoethanol breaks disulfide bonds in globular proteins.
Glycerol-high density solution- pull DNA sample and settle to the bottom of the well.
The electrophoretic migration rate through a gel is therefore determined only by the size of the protein.
All proteins, because of the negative charge imparted by SDS, move toward the positive electrode.
Molecular weights are determined by simultaneously running marker proteins of known molecular weight.
Native PAGE separates proteins based on their charge, mass, and shape (conformation), without denaturing them.
Isoelectric focusing (IEF) is an electrophoretic separation technique based on the isoelectric points (pI) of molecules.
Proteins are loaded; they will move to anode/cathode according to their net charge.
They will reach a point where the gel pH is equal to their pI.
Because proteins have zero net charge when pH = pI; they cease moving.
The protein is said to 'focus' at this point.

A 2-D gel electrophoresis technique is used to separate protein mixtures using two different principles, which improves resolution.
Western blotting is a method for separating and detecting specific proteins from a mixture of proteins.
The main steps are sample preparation and electrophoresis, electrophoretic transfer, blocking, antibody incubation, and detection.
ELISA (enzyme-linked immunosorbent assay) is an immunoassay detecting and quantifying small molecules, like antibodies, peptides, antigens or proteins.
ELISA is a simple and sensitive technique for quantitative information output, using enzyme-conjugated antibodies or fluorescence-conjugated antibodies.
ELISA relies on several key reagents, including antigen, antibody, enzyme-conjugated antibody, and a substrate.
Polymerase chain reaction (PCR) is a technique for amplifying target DNA sequences.
A thermal cycler is a laboratory instrument used to increase and decrease the temperature of the reaction mixtures in controlled, pre-programmed steps.
PCR requires several chemicals including template DNA, primers (forward and reverse), deoxynucleotide triphosphates (dNTPs), DNA polymerase, PCR buffers, and nuclease-free water.
Types of PCR: Conventional, Real-time, and Digital.
Real-time PCR allows real-time monitoring of the newly generated PCR product during the process by using fluorescence labelled oligonucleotide
DNA ladder is used as a molecular marker, which is a set of DNA standards with known base pair sizes for identifying the approximate size of the DNA sample during electrophoresis.
DNA amplified is visible using fluorescent dye, like Ethidium bromide.
Capillary electrophoresis is a method of separating charged macromolecules by their charge-to-mass ratio
Capillary electrophoresis also refers to the time required for molecules to travel through a matrix.

### Other Techniques

Some additional scientific techniques include (but are not limited to) the following: oligonucleotide/peptide synthesis, microarray (DNA or protein) and centrifuge.

### Centrifuge 1

A centrifuge is a device to separate particles from a solution by size, shape and density.
The principle of centrifugation is based on the centrifugal force generated during high-speed spinning that makes particles, with a greater density, to settle towards the bottom of a solution faster.
Relative centrifugal force (RCF) is a transferable unit used across manufacturers’ centrifuges.
Nomograms translate revolutions per minute (RPM) to relative centrifugal force (RCF) or vice versa.

### Centrifuge 2

Types of centrifugation methods include:
Cell fractionation, involving the disruption and lysis of a cell membrane via homogenization
Differential centrifugation, isolating different cellular components by separating them via successive cycles of high-speed centrifugation,
Gradient centrifugation, separating substances based on their density and sedimentation rate in a density gradient

Microscopy 1

A microscope is a laboratory instrument used to examine small objects or specimens not viewable by the naked eye.
The history of the microscope traces back to the Dutch Janssen brothers who invented the first microscope in 1590, further developed by Antonie van Leeuwenhoek
Types of microscopes
Light Microscopy (includes simple and compound microscopes, brightfield, darkfield, phase contrast)
Electron Microscopy (transmission and scanning)

Microscopy 2

Types of Light Microscopes: These include Simple, Compound, Bright Field, Dark Field, Phase Contrast, Confocal, and Polarized.
Working principles, components, and functions for each type.
Applications & Advantages
Limits & Usefulness