lecture 3 Practical applications of molecular biology Flashcards
what is a REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR)
*To use PCR to obtain a cDNA clone of a gene, total mRNA is first purified from cells.
*The first primer is added to the population of mRNAs, and reverse transcriptase is used to make a DNA strand complementary to the specific RNA sequence of interest.
*The second primer is then added, and the DNA molecule is amplified through many cycles of PCR.
what is QUANTITATIVE RT-PCR
Expression of individual genes can be measured using Quantitative RT-PCR
*Dyes added to the PCR fluoresce only when bound to dsDNA - used to track the progress of the reaction and deduce starting conc. of the mRNA being amplified.
*Fast and simple to perform.
*Preferred method for accurate measurement of mRNA levels.
*Used to detect rare viral RNAs to determine if a person is infected
how can PCR can be used to detect the presence of a pathogen in a clinical sample
- nasal sample is collected
- rare Sars-cov-2 virus in sample from an infected person is detected
- DNA is extracted
- reverse transcription and PCR amplification of Sars-cov-2 cDNA occurs
- a control is done using a sample from a non-infected person and compared using gel electrophoresis
how are Next-generation sequencing (NGS) methods are used for RNA analysis too – RNA-seq
*When reverse transcriptase is used to copy all RNAs into cDNAs, which are then fragmented and sequenced by NGS methods, this global analysis of mRNAs by RNA-seq provides a snapshot of gene expression.
*More abundant RNAs will have more cDNA copies, resulting in higher numbers of “sequence reads” for those RNAs.
*RNA-seq does not simply identify the RNAs in a sample but also provides information about their relative abundance.
how can we be PURIFYING PROTEINS
PURIFYING PROTEINS*Proteins can be released from bacteria by lysis and precipitation with ammonium sulphate
*Proteins can then be further separated by:
*Chromatography
ImmunoprecipitationSDS polyacrylamide-gel electrophoresis (SDS-PAGE)
*Two-dimensional gel electrophoresis
what is CHROMATOGRAPHY
CHROMATOGRAPHY*The sample is applied to the top of a cylindrical glass or plastic column filled with a permeable gel matrix, such as cellulose.
*A large amount of solvent is then passed slowly through the column and collected in separate tubes as it emerges from the bottom.
*As various components of the sample travel at different rates through the column, they are fractionated into different tubes
what are the Three types of matrices used for chromatography
A- ion-exchange chromatography
B-Gel-filtration chromatography
C- Affinity-chromatography
what is ion-exchange chromatography
The insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge.
*Matrices used for separating proteins include diethylaminoethylcellulose (DEAE-cellulose = positively charged), and carboxymethylcellulose (CM-cellulose = negatively charged).
*Matrices based on agarose or other polymers are also used.
*Strength of association depends on both ionic strength and pH of the solution passing down the column.
what is Gel-filtration chromatography
*Small inert but porous beads form the matrix.
*Molecules small enough to penetrate the matrix beads are delayed and travel more slowly through the column than larger molecules that can’t penetrate.
*Beads of cross-linked polysaccharide (dextran, agarose, or acrylamide) are available commercially in a wide range of pore sizes.
what is Affinity-chromatography
Affinity chromatography uses an insoluble matrix covalently linked to a specific ligand, such as an antibody or enzyme substrate, that will bind a specific protein.
*Enzyme molecules that bind to immobilised substrates can be eluted with a concentrated solution of the free form of the substrate molecule.
*Molecules that bind to immobilised antibodies can be eluted by dissociating the antibody–antigen complex with concentrated salt solutions or solutions of high or low pH.
*High degrees of purification can be achieved in a single pass through an affinity column.
what three different chromatographic steps can be used in succession to purify a protein
1- ion-exchange chromatography
2-Gel-filtration chromatography
3- Affinity-chromatography
what is IMMUNOPRECIPITATION
Immunoprecipitation is a rapid affinity purification method
*A useful alternative to affinity chromatography.
*Specific antibodies that recognise the protein to be purified are attached to small agarose beads.
*Rather than being packed into a column, as in affinity chromatography, a small quantity of the antibody-coated beads is simply added to a protein extract in a test tube and mixed in suspension for a short period of time —thereby allowing the antibodies to bind the desired protein.
*The beads are then collected by low-speed centrifugation, and the unbound proteins in the supernatant are discarded.
*Commonly used to purify small amounts of enzymes from cell extracts for analysis of enzymatic activity or for identification of associated proteins
how can we be ANALYSING PROTEINS
*Proteins can be separated by SDS polyacrylamide-gel electrophoresis (SDS-PAGE)
*Two-dimensional gel electrophoresis provides greater protein separation
*Specific proteins can be detected by blotting with antibodies – Western blotting
*Optical methods can monitor protein interactions
*Mass Spectrometry provides a highly sensitive method for identifying unknown proteins
*Protein structure can be determined using X-ray diffraction or nuclear magnetic resonance (NMR)
*Protein sequence and structure provide clues about protein function
what is An electrophoresis apparatus
*A polyacrylamide gel is sandwiched between two glass plates, with each end of the gel immersed in a buffer connected to an electrode.
*Think of an upright version of agarose DNA electrophoresis from the first lecture
What is SDS needed for?
*The detergent sodium dodecyl sulfate (SDS) and the reducing agent b-mercaptoethanol are used to solubilise the proteins.
*Individual polypeptide chains form a complex with negatively charged molecules of SDS.
*They migrate through the gel toward the anode.
*A smaller polypeptides move more quickly through the gel this technique is used to determine the approximate mass of a polypeptide chain.
*Carbohydrate or phosphorylation can cause small changes in a protein’s migration in the gel, giving an incorrect apparent mass estimate
what does Analysis of protein samples by SDS polyacrylamide-gel electrophoresis entail?
*The photograph shows a Coomassie blue–stained gel that has been used to detect the proteins present at successive stages in the purification of an enzyme.
*The leftmost lane (lane 1) contains the complex mixture of proteins in the starting cell extract.
*Each succeeding lane analyses the proteins obtained after a chromatographic fractionation of the protein sample analysed in the previous lane.
*The same amount of protein (10 μg) was loaded onto the gel at the top of each lane
what are the TWO-DIMENSIONAL GEL ELECTROPHORESIS
*Two-dimensional gel electrophoresis provides greater protein separation than one-dimensional SDS-PAGE.
*Works on the principle of isoelectric focusing followed by separation according to mass.
how is Separation of protein molecules by isoelectric focusing different depending on pH?
At low pH (high proton concentration), the carboxylic acid groups of proteins tend to be uncharged (–COOH) and their nitrogen-containing basic groups fully charged (–NH3+), giving most proteins a net positive charge.
At high pH (low proton concentration), the carboxylic acid groups are negatively charged (–COO–) and the basic groups tend to be uncharged (–NH2), giving most proteins a net negative charge.
Between the extremes of pH, proteins have a mixture of negative and positive charge.
what is the isoelectric point?
At the isoelectric point, the protein has no net charge.
Thus, when a tube containing a fixed pH gradient is subjected to a strong electric field in the appropriate direction, each protein species migrates until it forms a sharp band at its isoelectric point.
In this case the isoelectric pH is 6.5.
what does western blotting do?
Specific proteins can be detected by blotting with antibodies
*Proteins are separated on a polyacrylamide gel.
*The gel is stained with Coomassie blue to reveal the most abundant proteins (A).
*The proteins in the gel are transferred to a membrane and exposed to antibodies directed against a specific protein.
*Unbound antibodies are washed away, and antibodies bound to the protein were detected with a fluorescent label (B).
*Small amounts of a single rare protein can be detected in a complex mixture of other proteins.
what is MASS SPECTROMETRY (MS)
MS is a highly sensitive method for identifying unknown proteins
*Contain an ion source that generates gaseous peptides under conditions that render most molecules positively charged.
*Ions are accelerated into a mass analyser, which separates the ions based on their mass and charge
how can we perform Measurement of binding with fluorescence anisotropy
Low anisotropy- depolarized emission
High anisotropy- polarized emission
what is X-RAY DIFFRACTION -
A narrow beam of x-rays is directed at a protein crystal
The atoms in the crystal scatter some of the beam, and the scattered waves reinforce one another at certain points and appear as a pattern of diffraction spots
The diffraction pattern, together with the amino acid sequence of the protein, can be used to produce an atomic model
A simplified version of the model shows the protein’s main structural features more clearly:α helices, greenβ strands, red
As for DNA, a BLAST search can be made of an amino acid sequence what do the red, yellow and green colours represent?
Green blocks indicate differences in sequence.
Yellow bars summarise the similarities: when the two amino acid sequences are identical, the residue is shown; similar amino acid substitutions are indicated by a plus sign (+).
Only one small gap has been introduced—indicated by the red arrow headat position 194 in the Query sequence—to align the two sequences maximally
