Analytical techniques (Protein and DNA) Flashcards
1
Q
restriction enzyme
A
- Endonuclease enzymes that recognize specific base sequences in double-stranded DNA
- Cleave both strands of the duplex at specific places.
3
Q
Southern blotting
A
- A technique used to locate and identify a DNA fragment containing a specific sequence;
- a mixture of fragments is separated by electrophoresis,
- transferred to a nitrocellulose sheet,
- hybridized to a radioactively labeled DNA probe complementary to the desired sequence,
- Visualized by autoradiography.
4
Q
DNA probe
A
- A radioactively labeled, single-stranded specific base sequence
- Used to locate a complementary sequence among DNA fragments displayed on an electrophoretic gel.
5
Q
controlled termination of replication (Sanger dideoxy method)
A
- A DNA-sequencing technique that employs controlled interruption of enzymatic replication of the molecule to be analyzed.
- DNA polymerase I is used with a primer,
- the four deoxynucleoside triphosphates, and
- a 2’, 3’-dideoxy analog of one of them.
- Fragments of various lengths are produced in which the dideoxy analog is at the 3’ end.
- Four sets of chain-terminated fragments (one for each analog) are then displayed by electrophoresis and autoradiography
- the base sequence can be read from the four lanes of the gel.
6
Q
polymerase chain reaction (PCR)
A
- A method for amplifying DNA sequences using DNA polymerase;
- a series of three-step cycles is employed,
- in which parental DNA strands are separated by heating,
- primers to flanking regions of the target sequence are annealed to the separated strands,
- the primers are then extended by DNA synthesis.
8
Q
plasmid
A
- Circular duplex DNA molecules that replicate autonomously
- act as accessory chromosomes in bacteria;
- they carry useful genes but are disposable under certain conditions.
9
Q
cloning vector
A
- These are plasmids or bacteriophage that allow the insertion and replication of DNA fragments into bacteria for the purpose of cloning.
- They often feature a polylinker region that includes many unique restriction sites within its sequence,
- allowing the region to be cleaved with many different restriction enzymes or combinations of enzymes.
- This provides great versatility in the DNA fragments that can be inserted.
10
Q
reporter gene
A
- Genes which encode readily-detectable markers such as:
- antibiotic-resistance enzymes
- fluorescent proteins.
11
Q
northern blotting
A
- A technique analogous to Southern blotting,
- A mixture of RNA fragments is separated by electrophoresis,
- transferred to a nitrocellulose sheet,
- hybridized to a radioactively labeled DNA probe complementary to the desired sequence,
- visualized by autoradiography.
- The technique can therefore be used to locate and identify an RNA fragment containing a specific sequence.
12
Q
lambda (λ) phage
A
- A bacteriophage cloning vector that can be incorporated into the hosts genome
- Thus can be replicated indefinitely
- Or can be expressed and destroy the host.
14
Q
yeast artificial chromosome (YAC)
A
- A DNA molecule that can be used to clone DNA inserts ranging from 100 to 1000 kb in length;
- these molecules contain a centromere,
- an autonomously replicating sequence,
- a pair of telomeres,
- selectable marker genes,
- an insertion site for the sequence to be cloned.
15
Q
genomic library
A
- A collection of DNA fragments,
- inserted into vector molecules,
- Represents the entire genome of an organism.
16
Q
complementary DNA (cDNA)
A
- DNA complementary to an mRNA sequence.
18
Q
site-directed mutagenesis
A
- A method in which a primer containing a mismatched nucleotide is used to produce a desired change in a DNA sequence.
- It can readily produce mutant proteins with single amino acid substitutions.
19
Q
cassette mutagenesis
A
- A means of introducing a variety of mutations into a gene of interest.
- A short segment of plasmid harboring the original gene is removed by restriction enzyme treatment.
- A synthetic double-stranded oligonucleotide (the cassette) carrying the genetic alterations of interest is subsequently inserted.
20
Q
next-generation sequencing
3 examples of it:
A
- Platforms that enable the rapid determination of a complete genome sequence
- Combining breakthroughs in the handling of very small amounts of liquid,
- high-resolution optics,
- computing power.
- Examples:
- Pyrosequencing
- ion semiconductor sequencing
- reverse terminator sequencing
21
Q
quantitative PCR (qPCR)
A
- A polymerase chain reaction-based technique
- For determining the amount of individual mRNA molecules present in a population of RNA molecules.
22
Q
DNA microarray (gene chip)
A
- A solid support such as a microscope slide to which are affixed oligonucleotides or cDNAs corresponding to specific genes.
- Fluorescently labeled cDNA is hybridized to the slide
- Reveals the expression level for each gene,
- Identifiable by its known position within the microarray.
23
Q
vector
A
- A DNA molecule that can replicate autonomously in an appropriate host organism.
- Vectors are designed to enable the rapid, covalent insertion of DNA fragments of interest.
25
Q
genome editing
A
- Highly specific modification of genomic DNA.
26
Q
RNA interference
A
- The suppression of the transcription of a gene
- following the introduction into the cell of double stranded RNA molecule that contained sequences present in the suppressed gene.
28
Q
tumor-inducing plasmid (Ti plasmid)
A
- Plasmids carried by A. tumefaciens
- Encode instructions for induction of tumor state in infected plants cells.
- The resulting tumor tissue is called a crown gall.
30
Q
expression vector
A
- A plasmid-cloning vector
- Has been optimized for the expression of large amounts of recombinant protein encoded by the vector.
32
Q
bacterial artificial chromosome (BAC)
A
- An artificial bacterial chromosome,
- a highly engineered version of the E. coli fertility (F factor), that can serve as a cloning vector for inserts as larges as 300 kb.
36
Q
cDNA library
A
- A collection of all of the complementary DNA for all mRNA that a cell contains,
- which have been inserted into vectors,
- then inserted into bacteria.
42
Q
transgenic mouse
A
- A mouse that harbors a foreign gene.
- Transgenic mice are a powerful means of exploring the role of a specific gene in the following in an organism:
- development,
- growth, and
- behavior
43
Q
gene disruption (gene knockout)
A
- Inactivating a gene
- Looking for resulting abnormalities in order to determine the gene’s function.
46
Q
RNA-induced silencing complex (RISC)
A
- An assembly of specific proteins that facilitate the process of RNA interference.
48
Q
gene gun (bombardment-mediated transformation)
A
- A means of transforming plant cells.
- DNA is coated onto 1-mm-diameter tungsten pellets,
- These microprojectiles are fired at the target cells with a velocity greater than 400 m s–1.
49
Q
Salting out
A
- A protein purification technique
- based on the fact that the solubility of most proteins is lowered at higher salt concentrations.
- Consequently, different proteins will precipitate at varying salt concentrations.
50
Q
dialysis
A
- The process of removing small molecules from a solution containing a mixture of large molecules and small molecules.
- The mixture is placed in a bag made of a semipermeable membrane,
- Is then placed in a different solution.
- The membrane allows escape by the small molecules but not the large molecules.
51
Q
gel-filtration chromatography
A
- A separation technique based on size differences.
- A sample is applied to a column consisting of porous beads.
- Large molecules move through the column faster because they cannot enter the beads and, thus, have a shorter path to travel.
52
Q
ion-exchange chromatography
A
- A protein purification technique that relies on the charge of proteins.
- Proteins are applied to an inert matrix to which is attached a charged moiety (e.g., a carboxylate group).
- Proteins will bind to the matrix with an affinity proportional to their content of the counterion (i.e., positive charges in the case of the carboxylate matrix).
55
Q
affinity chromatography
A
- A protein purification technique based on the high affinity many proteins have for specific chemical groups.
- Such groups are attached to an inert matrix,
- The protein sample is applied;
- only those with an affinity for the groups will bind.
56
Q
high-performance liquid chromatography (HPLC)
A
- A column chromatography technique in which the column materials are very finely divided
- As a consequence, possess more interaction sites and thus greater resolving power.
- Because the column is made of finer material, pressure must be applied to the column to obtain adequate flow rates.
- The net result is both high resolution and rapid separation.
58
Q
isoelectric focusing
A
- A technique for separating proteins.
- A mixture of proteins is electrophoresed in a pH gradient;
- each protein will migrate in the electrical field until it reaches its isoelectric point.
59
Q
two-dimensional electrophoresis
A
- A means of analyzing a protein sample
- in which the sample is initially fractionated in one dimension by isoelectric focussing,
- and subsequently fractionated in a second dimension, perpendicular to the first, by SDS-polyacrylamide gel electrophoresis.
60
Q
enzyme-linked immunosorbent assay (ELISA)
A
- An assay for quantifying the presence of an antigen
- Uses an enzyme linked to an antibody to the antigen.
61
Q
western blotting
A
- An immunoassay technique used to detect a specific protein in a cell or in body fluid.
- A sample is electrophoresed in an SDS-polyacrylamide gel,
- the resolved proteins are transferred to a polymer sheet,
- Tthen an antibody specific for the protein of interest is incubated with the blotted sample;
- other antibodies or radioactive markers may then be used to help visualize the desired antigen-antibody complex.
62
Q
cation exchange
A
- Ion-exchange chromatography
- A protein mixture is passed through a column containing a matrix bearing negative charges.
- Proteins bearing positive charges will bind to the column
- those with negative charges will pass through the column.
63
Q
matrix-assisted laser desorption/ionization (MALDI)
A
- A technique for determining a proteins mass.
- The protein is evaporated to dryness in the presence of a volatile, aromatic compound that can absorb light at specific wavelengths.
- A laser pulse excites and vaporizes the matrix, converting some of the protein into the gas phase.
- Subsequent collisions enable the intermolecular transfer of charge, ionizing the protein.
- The newly formed ions then enter the mass analyzer,
- They are distinguished on the basis of their mass-to-charge ratios.
64
Q
anion exchange
A
- Ion-exchange chromatography
- A protein mixture is passed through a column containing a matrix bearing positive charges.
- Proteins bearing negative charges will bind to the column
- those with positive charges will pass through the column.
65
Q
time-of-flight (TOF) mass analyzer
A
- A mass analyzer used in conjunction with matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI).
- The time-of-flight (TOF) analyzer accelerates the ions generated by MALDI or ESI through a chamber under a fixed electrostatic potential.
- The mass of each ion can be determined by measuring the time required for each ion to pass through the chamber.
66
Q
Edman degradation
A
- used in sequencing proteins.
- Sequential removal of the N-terminal amino acid from a protein as a phenylthiohydantoin derivative;
67
Q
tandem mass spectrometry
A
- The utilization of two mass analyzers to determine protein sequence.
- Ions of proteins that have been analyzed by a mass spectrometer are broken into smaller peptide chains by bombardment with atoms of an inert gas such as helium or argon.
- These new fragments can be passed through a second mass analyzer for further mass characterization.
68
Q
overlap peptide
A
- Peptides resulting from degradation of a protein by two different procedures that are subsequently sequenced.
- The sequence of a peptide from one degradation procedure frequently overlaps the sequences of two or more peptides of the other degradation procedure,
- thereby establishing the order of the peptides.
70
Q
solid-phase method
A
- A means of synthesizing discrete peptides
- in which amino acids are added step-by-step to a growing peptide chain that is anchored to an insoluble matrix.
71
Q
x-ray crystallography
A
- A technique to determine the three-dimensional structure of protein crystals at atomic resolution
- by examining the diffraction pattern of x-rays striking the crystal.
72
Q
nuclear magnetic resonance (NMR) spectroscopy
A
- Means of determining the structure of a protein in solution
- based on the ability of certain atoms in a protein to absorb electromagnetic radiation.
74
Q
gel electrophoresis
A
- A technique used to separate charged molecules,
- such as proteins and nucleic acids, which is
- based on the fact that such molecules will move at differing rates through a gelatinous material,
- such as polyacrylamide or agarose, when subjected to an electric field.
- Separation depends on factors such as:
- net charge,
- size,
- shape of the molecules.
79
Q
fluorescence microscopy
A
- An optical microscope capable viewing materials by reflection and absorption
- as well as visualizing fluorescent materials.
81
Q
electrospray ionization (ESI)
A
- A mass spectrometry technique that allows the analysis of proteins.
- A solution of the protein is passed through an electrically charged nozzle.
- Droplets of the protein, now charged, emerge from the nozzle into a chamber of very low pressure,
- evaporating the solvent and ultimately yielding the ionized protein.
- The newly formed protein ions then enter the mass analyzer,
- where they are distinguished on the basis of their mass-to-charge ratios.
86
Q
peptide mass fingerprinting
A
- A technique for protein identification that involves cleaving a protein by chemical or enzymatic methods
- followed by chromatographic separation and mass spectrometry.
