Restriction Enzyme Star Activity Factors

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What is the main reason that restriction enzymes may exhibit 'star activity' and cut at sequences other than their expected recognition sequence?

The enzymes are inherently less specific under non-standard conditions. (correct)

The enzymes have a divalent cation imbalance.

The enzymes are exposed to prolonged reaction times.

The enzymes are contaminated with organic solvents.

What is the significance of Restriction Fragment Length Polymorphisms (RFLPs)?

RFLPs are used to detect structural changes in chromosomes associated with disease.

RFLPs are the basis for the first molecular-based human identification and mapping methods.

RFLPs are used to analyze the size and number of restriction fragments in an individual's DNA.

All of the above. (correct)

Which of the following is NOT a condition that can induce 'star activity' in restriction enzymes?

Suboptimal buffer conditions.

Prolonged incubation at low temperature. (correct)

pH greater than 8.0.

High concentration of enzymes.

What is the main purpose of procedures performed in the clinical molecular laboratory?

To visualize or detect a particular gene or region of DNA. Signup and view all the answers

What is the significance of the Southern blot technique?

It was the first method that did not require cloning to analyze DNA. Signup and view all the answers

Which of the following can contribute to differences in the number and location of restriction sites for a given restriction enzyme in human DNA?

Genetic variations between individuals. Signup and view all the answers

What is the primary purpose of the depurination step when preparing larger DNA fragments for blotting?

To increase the efficiency of denaturing the larger DNA fragments Signup and view all the answers

What is the primary indication that the restriction enzyme digest was incomplete based on the information provided?

A large aggregate of DNA near the top of the lane Signup and view all the answers

What is the recommended voltage and run time for resolving 10,000- to 20,000-bp DNA fragments in a 0.7% agarose gel?

20 amperes for 16 hours Signup and view all the answers

What is the purpose of staining the agarose gel with ethidium bromide and illuminating it with UV light?

To visualize the resolved DNA fragments Signup and view all the answers

What is the primary indication that the DNA sample is degraded based on the information provided?

A smear located primarily in the lower region of the lane Signup and view all the answers

What is the purpose of the blotting (transfer) step after the DNA fragments have been resolved by electrophoresis?

To allow hybridization of the DNA fragments with a complementary probe Signup and view all the answers

What does sequence complexity refer to?

The length of nonrepetitive nucleotide sequences Signup and view all the answers

What does Cot 1/2 represent in nucleic acid sequences?

The time required for half of a sequence to anneal Signup and view all the answers

How do longer probes differ from shorter probes in terms of specificity?

Longer probes have higher specificity due to point mutations Signup and view all the answers

Why are short probes ideal for mutation analysis?

Short probes have high binding affinity towards mutations Signup and view all the answers

What is the effect of increased probe concentration on hybridization sensitivity?

Increases sensitivity Signup and view all the answers

Where is hybridization generally performed?

In hybridization bags or on glass slides Signup and view all the answers

What is the recommended volume of hybridization buffer for 100 cm2 of membrane surface area?

10 mL Signup and view all the answers

What is the primary function of formamide in the hybridization buffer?

To decrease the optimal hybridization temperature Signup and view all the answers

Which method is recommended for maintaining a constant temperature during hybridization?

Both a) and c) Signup and view all the answers

What is the optimal hybridization time for short probes (less than 20 bases)?

1 to 2 hours Signup and view all the answers

Which polymers can accelerate the hybridization rates for probes longer than 250 bases?

Dextran sulfate, polyethylene glycol, or polyacrylic acid Signup and view all the answers

What is the optimal hybridization temperature for a double-stranded DNA probe?

Tm - 25°C Signup and view all the answers

Which of the following is a key characteristic of bead array systems used for clinical tests?

The beads are coated with a unique fluorescent dye to identify different targets Signup and view all the answers

What is the main advantage of solution hybridization compared to other hybridization techniques?

It allows for the detection of low amounts of target RNA Signup and view all the answers

What key technological advancement allowed microarrays to become more versatile compared to macroarrays?

All of the above Signup and view all the answers

Which of the following is a common application of bead array systems in clinical testing?

Screening for infectious diseases Signup and view all the answers

What is the key difference between solution hybridization and microarray techniques in terms of target and probe immobilization?

In solution hybridization, neither the probe nor the target is immobilized, while in microarrays, both the probe and the target are immobilized Signup and view all the answers

What is the main advantage of microarrays compared to macroarrays in terms of the number of targets that can be screened simultaneously?

Microarrays can screen up to 80,000 targets simultaneously, while macroarrays are limited to 10,000 targets Signup and view all the answers