Analysis and Characterization of Nucleic Acids and Proteins

Question 1

CRSPR

Answer 1

Clustered Regularly Interspaced Short Palindromic Repeats. Classes of repeated DNA sequences that provide bacterial immunity to bacteriophage or exogenous plasmids.

Question 2

crRNA

Answer 2

CRSPR RNA. Short, mature RNA sequences transcribed from the CRSPR spacer regions, processed from pre-crRNA carrying complementary sequences to a target DNA.

Question 3

tracrRNA

Answer 3

Trans-activating CRSPR RNA. noncoding RNA that forms a complex with a crRNA and cas9 endonuclease in bacteria to target invading DNA.

Question 4

Southern Blot

Answer 4

A method used to analyze specific regions of DNA in a mixture of other DNA regions. Genomic DNA is isolated and cut with restriction enzymes. The fragments are separated by gel electrophoresis, denatured, and then transferred to a solid support such as nitrocellulose. They are then exposed to a labeled probe that is complementary to the region of interest. The signal of probe is detected to indicate the presence or absence of the sequence in question.

Question 5

Nitrocellulose

Answer 5

A solid support used in a southern blot with high binding capacity for nucleic acids and proteins.

Question 6

Probe

Answer 6

Complementary DNA or RNA. Single stranded fragment. Can be a nucleic acid or protein with a detectable signal that specifically binds to the complementary sequences or target protein.

Question 7

Depurination

Answer 7

Removal of purines from the sugar-phosphate backbone. Process where the gel is soaked in dilute hydrogen chloride solution, where it removed purine bases from the sugar phosphate backbone. This is done for large fragments prior to denaturation.

Question 8

Denaturation for blotting

Answer 8

DNA is denatured by exposing the gel to sodium hydroxide.

Question 9

Blotting

Answer 9

The denatured DNA is is transferred to a solid substrate that will facilitate probe binding and signal detection.

Question 10

Capillary Transfer

Answer 10

Movement of nucleic acid or protein from a gel matrix to a membrane by capillary action. Simple and inexpensive. The gel is placed on top of a reservoir of buffer, which can be a shallow container or filter papers soaked in high salt buffer or transfer buffer. The nitrocellulose membrane is places on the gel, and dry absorbent filter paper or paper towels are stacked on top of the membrane. The buffer will move from the top to the bottom by capillary action. The movement of the buffer through the gel will carry the DNA out of the gel and bind to the nitrocellulose membrane. There can be a loss of information or staining if there are bubbles, salt crystals, or other particles between the gel and the membrane. Slow. Can take a few hours to overnight for large fragments.

Question 11

Electrophoretic Transfer

Answer 11

Uses electric current to move nucleic acid or protein from the gel matrix to the membrane. Uses electrodes attached to membranes above and below the gel. Takes 2-3 hours. More expensive and maintenance of the equipment.

Question 12

Vacuum Transfer

Answer 12

Uses suction to move the nucleic acid or protein from the gel matrix to the membrane in a recirculating buffer. Takes 2-3 hours. More expensive and maintenance of the equipment.

Question 13

Prehybridization

Answer 13

Treatment of membranes before introduction of the probe to minimize nonspecific binding. Incubating the membrane in the same buffer in which the probe will subsequently be introduced or in a specially formulated prehybridization buffer solution. The membrane is exposed to this for 30 minutes to several hours. The sample is then ready for hybridization of the probe.

Question 14

Northern Blot

Answer 14

Modification of the southern blot. Investigates RNA structure and quality. Used to analyze specific RNA transcripts.

Question 15

Western Blot

Answer 15

Modification of the southern blot. The immobilized target is protein or peptides.

Question 16

Denaturing Gels

Answer 16

A polyacrylamide gel containing a substance that well denature DNA.

Question 17

Epitopes

Answer 17

Specific antigenic sites on the protein.

Question 18

Primary Antibody

Answer 18

Antibody that directly binds a target molecule.

Question 19

Blocking

Answer 19

Covering nonspecific binding site before probe hybridization.

Question 20

Peptide Nucleic Acids

Answer 20

PNA. Can be used as probes. Synthetic. Nucleic acid with peptide bonds replacing the sugar-phosphate backbone.

Question 21

Locked Nucleic Acids

Answer 21

LNA. Can be used as probes. Synthetic. Nucleic acid with modified sugar-phosphate backbone.

Question 22

Secondary Antibody

Answer 22

Antibody that binds primary antibody to a target molecule.

Question 23

Haptens

Answer 23

Small molecules that attach to protein carriers, carbohydrates, nucleic acids, and whole cells and tissue extracts can be used to generate an antibody response.

Question 24

Probe Labeling

Answer 24

End labeling, nick translation, and random priming.

Question 25

Polyclonal Antibodies

Answer 25

Products of a generalized response to a specific antigen or epitope, usually a peptide or protein. Produced in vivo.

Question 26

Nick Translation

Answer 26

A type of probe labeling where the labeled nucleotides are incorporated into single-stranded breaks, or nicks, that are substrates for nucleotide addition by DNA polymerase.

Question 27

Monoclonal Antibodies

Answer 27

Antibody preparation designed to recognize a defined antigen or epitope.

Question 28

Hybridomas

Answer 28

Hybrid cells that could grow in culture and secrete antibodies, used to make monoclonal antibodies.

Question 29

Biotin

Answer 29

A vitamin that is used in the laboratory for nonradioactive probe detection.

Question 30

Digoxygenin

Answer 30

A steroidal compound used for nonradioactive probe detection.

Question 31

Dot Blots

Answer 31

The target is deposited in a circle or dot. Useful for multiple qualitative analyses where many targets are being compared, such as in mutational screening.

Question 32

End Labeling

Answer 32

A type of probe labeling where radioactive or other labeled nucleotides are added to the end of a nucleic acid using terminal transferase.

Question 33

Terminal Transferase

Answer 33

An enzyme that adds nucleotides to the end of a strand of nucleic acid without using a template.

Question 34

Nick Translation for probes

Answer 34

A type of probe labeling where the labeled nucleotides are incorporated into single-stranded breaks, or nicks, that are substrates for nucleotide addition by DNA polymerase.

Question 35

Random Priming

Answer 35

A type of probe labeling where it generates new single-stranded versions of DNA with the incorporation of the labeled nucleotides.

Question 36

Reverse Dot Blots

Answer 36

Many different unlabeled probes are immobilized on the membrane and the test sample is labeled for hybridization with the immobilized probes.

Question 37

C0T1/2

Answer 37

The time required for half of a double-stranded sequence to anneal under a given set of conditions.

Question 38

Dot Blots

Answer 38

Hybridization technique where multiple targets are deposited in a circle or dot on a membrane all exposed to the same probe. Useful for multiple qualitative analyses where many targets are being compared, such as in mutational screening.

Question 39

Slot Blots

Answer 39

The targets are deposited in an oblong bar on a membrane instead of dots. More accurate for quantification by densitometry scanning because they eliminate the error that may arise from scanning thru a circular target.

Question 40

Genomic Array Technology

Answer 40

A type of hybridization analysis allowing the simultaneous study of large numbers of targets. Arrays are applied to gene amplification or deletion on comparative genome hybridization arrays and to gene-expression analysis on expression arrays. Macroarrays, microarrays, high-density oligonucleotide arrays, microelectronic arrays.

Question 41

Macroarrays

Answer 41

A membrane containing multiple immobilized probes.
Reverse dot blots of up to several thousand targets on nitrocellulose membranes. Limited to the area of the membrane and the specimen requirements.

Question 42

Gel Mobility Shift Assay

Answer 42

Electrophoretic mobility shift assay. Looks at protein-DNA and protein-RNA interactions. After mixing the labeled DNA with the test material, a change in mobility indicates binding of a component in the test material to the probe protein or nucleic acid. Used to identify trans factors that bind to cis-acting elements that control gene regulation.

Question 43

Phosphor

Answer 43

A substance that glows after exposure to electrons or UV light.

Question 44

Microarrays

Answer 44

A small glass slide or other substrate containing multiple immobilized probes.

Question 45

High-density Oligonucleotide Arrays

Answer 45

A large number of probes, more than 100,000, synthesized in place on the substrate. Used for mutation and polymorphism analysis, DNA methylation, and sequencing.

Question 46

Solution Hybridization

Answer 46

Neither the probe nor the target is immobilized. Probes and targets bind in solution. Used to measure mRNA expression, especially when there are low levels of RNA.

Question 47

RNase Protection

Answer 47

A type of solution hybridization. S1 mapping. A method used to detect transcripts by hybridizing test RNA with a labeled probe and removing an un-hybridized target and probe with singe-strand specific nucleus. The labeled probe is hybridized to the target sample in solution.

Question 48

Antigen

Answer 48

A toxic or foreign substance that induces an immune response, the production of antibodies.