Question 1

The Gen-Probe AccuProbe system uses the following technology principle:

Accepted Answer

A single-stranded,chemiluminescent labeled-DNA probe is designed to hybridize to the target organism's ribosomal RNA (rRNA),forming a DNA:RNA duplex.A detector called a liminometer is used to detect these labeled duplexes.The liminometer gives a reading in relative light units (RLU),and the RLU result for a suspicious culture is compared to a positive cutoff RLU value: any reading over the cutoff value is positive,and readings below are negative.

Question 2

The variables that affect the outcome of a given hybridization reaction include all of the following EXCEPT:

Accepted Answer

These variables affecting the outcome of a given hybridization reaction include temperature,the nucleotide base composition of the probe,the length of the probe,the probe concentration,the degree of complementarity between the target and the probe,ionic strength (salt concentration),and pH.When one or more of these variables is not optimized for a hybridization assay,the hybrids may not efficiently form.

Question 3

Who received the Nobel Prize in Chemistry for inventing (polymerase chain reaction (PCR)?

Accepted Answer

The technique of synthetically synthesizing DNA was first described in 1971 by Khorana and colleagues,although Kary B.Mullins and others at the Cetus Corporation in California developed PCR into the current application in the early 1980s,eventually earning him the Nobel Prize in Chemistry for PCR.

Question 4

Many hybridization assay conditions are based on the expectation that a probe:

Accepted Answer

Many hybridization assay conditions are based on the expectation that a probe has exact or near-exact complementarity to the target nucleic acid.This will not always be the case.When conditions are stringent,exact matches between the probe and target will hybridize first,whereas mismatches form duplexes more slowly.

Question 5

The target nucleic acid strand is:

Accepted Answer

This is the portion of DNA that we are interested in identifying.The target nucleic acid molecule is typically either immobilized on a solid support mechanism or suspended in solution.

Question 6

What types of compounds are used for labeling probes?

Accepted Answer

Nonisotopic labels include biotin,digoxigenin (DIG),and fluorescein.

Question 7

The steps for performing the Northern blot test include all the following EXCEPT:

Accepted Answer

Northern blots may be used to determine the size of particular RNA molecules and to semiquantitate the amount of a particular RNA transcript.The actual procedure of the Northern blot is similar to that of the Southern blot; RNA is separated in an agarose gel,transferred to a membrane,immobilized,and detected with a probe that hybridizes to the RNA species of interest.Detection is also accomplished to the type of label on the probe.

Question 8

A Northern blot is used to detect:

Accepted Answer

A Northern blot is used to detect RNA molecules.

Question 9

The function of the probe is to:

Accepted Answer

The function of the probe is to form a duplex with every complementary sequence available in the reaction,and the probe must be suited for the particular hybridization reaction that will be used.

Question 10

Nucleic acid hybridization is:

Accepted Answer

Conceptually,nucleic acid hybridization refers to the formation of hydrogen bonds between nucleotides of single-stranded DNA or RNA molecules that are complementary.This forms a stable double-stranded nucleic acid molecule under the right conditions.

Question 11

The principle of solid support hybridization (often called blotting)is:

Accepted Answer

In solid support hybridization (a technique often called "blotting"),the target nucleic acid is transferred and immobilized to a membrane,composed of either nitrocellulose or nylon.The solid membrane is often pretreated to reduce nonspecific probe binding to the membrane itself.Labeled probe is then hybridized to the immobilized nucleic acid,and washing steps are utilized to remove excess probe and clarify the signal.

Question 12

The steps for performing the Southern blot test include all the following EXCEPT:

Accepted Answer

The DNA is first digested because chromosomal DNA is too large to separate in an agarose gel.Once the DNA has been separated and immobilized onto a solid membrane,labeled probe is hybridized to the specific target DNA sequence and detected.

Question 13

In a hybridization reaction,the pH affects:

Accepted Answer

pH affects the stability of double-stranded nucleic acid molecules in solution.An alkaline pH promotes dissociation of double-stranded molecules,whereas acidic pH solutions can depurinate probes and target nucleic acid molecules.

Question 14

The principle of in-solution hybridization is:

Accepted Answer

Several manufacturers have developed useful assays that promote hybridization between a labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells.Generally,detection methods for these commercial systems are chemiluminescent based.

Question 15

In situ hybridization,first described in 1969,is where:

Accepted Answer

In situ hybridization,first described in 1969 by Pardue and Gall,is also not often used in clinical microbiology laboratories.This is a method of hybridization where DNA or RNA transcript can be detected directly in tissue with labeled probes.This technique is often performed directly in tissue that has been embedded in paraffin.In situ hybridization can also detect nucleic acids in intact cells and chromosomal genetic material.

Question 16

The probe is:

Accepted Answer

The probe is usually a single-stranded DNA or RNA oligonucleotide that is labeled with an attached report chemical or radionucleotide that can be detected either visually,by film,or by an instrument.The probe is produced synthetically to detect a specific target nucleic acid sequence of a given microorganism.

Question 17

How is the stability of a given hybrid calculated?

Accepted Answer

The stability of a given hybrid can be calculated by determining the melting temperature of a probe.The melting temperature is the temperature at which 50% of the hybrids have formed and 50% of the single-stranded nucleic acid molecules are still dissociated.The melting temperature is dependent on the nucleotide composition of the probe.

Question 18

Uses of probes include all of the following EXCEPT:

Accepted Answer

Probes have many uses including the detection of microbial pathogens in many different types of samples,gene expression analysis,gene rearrangements and chromosomal translocations,point mutations,and other clinical diseases.

Question 19

The Southern blot test separates:

Accepted Answer

The Southern blot was first described in 1975 by E.M.Southern; he described a technique whereby chromosomal DNA is separated.

Question 20

How do higher probe concentrations affect a given hybridization reaction?

Accepted Answer

Higher probe concentrations typically lower the reaction time by saturating all of the available probe target sequences.However,excessive probe concentrations promote nonspecific binding of the probe to nontarget sequences.

Quiz 11: Applications of Molecular Diagnostics