Chapter 5: Manipulation of Nucleic Acids

Exonuclease

An enzyme that attacks the ends of molecules, removing nucleotides from the end of a DNA or RNA strand.

Endonuclease

An enzyme that cleaves the nucleic acid chain in the middle, producing fragments of different lengths.

Isoschizomer

Two restriction enzymes that share the same recognition sequence and cut site.

Neoschizomer

A subset of isoschizomers that recognize the same sequence but cleave at different positions.

Star activity

Imprecise or random cleavage of DNA by restriction enzymes that occurs under unnatural reaction conditions.

Sticky ends

Ends of a double-stranded DNA molecule that have unpaired single-stranded overhangs, generated by a staggered cut.

Restriction map

A diagram showing the location of cut sites on DNA for a variety of restriction enzymes.

RFLP

A difference in restriction enzyme sites between two related DNA molecules that result in restriction fragments of different lengths.

Autoradiography

A technique that uses photographic film on a gel or membrane to identify the exact location of radioactive DNA.

Scintillation counting

A method for detection and counting of individual microscopic pulses of light.

Southern blotting

A method to detect single-stranded DNA that has been transferred to a solid support using a probe that binds DNA.

Northern blotting

A hybridization technique in which a DNA probe binds to an RNA target molecule.

Western blotting

A detection technique in which a probe, usually an antibody, binds to a protein target molecule.

FISH

Using a fluorescent probe to visualize a molecule of DNA or RNA in its natural location.

What is the purpose of restriction enzymes in nature?

They evolved as a defense mechanism from foreign DNA by recognizing and cleaving specific DNA sequences.

Describe the naming system for restriction enzymes.

It identifies the bacterial species (strain) from which the enzyme was purified; first letter capitalized from Genus, next letters lower case from species, sometimes strain is represented, and Roman numeral indicates the number of restriction enzymes in the same species.

What are the two major classes of restriction enzymes?

Type I, which cuts DNA a thousand or more base pairs away and Type II, which cuts the DNA within the recognition site.

How is DNA concentration determined using a spectrophotometer?

By measuring the amount of ultraviolet light absorbed at 260nm, which is directly proportional to the concentration of DNA.

Interpret A260/280 and A230/260 readings.

A260/280 indicates ‘pure’ DNA with minimal protein contamination, and A230/260 shows minimal presence of residual chemicals like phenol or salts.

What is the phosphoramidite method of DNA synthesis?

A method for artificial synthesis of DNA that utilizes a reactive phosphoramidite group to link nucleotides.

• 3’ to 5’ synthesis

• 5’-OH blocked by dimethoxytrityl (DMT group)

• Phosphoramidite nucleotides (single phosphorus) - activates

What are the two radioactive isotopes used to label nucleic acids?

1. Phosphorus-32 (P-32) and 2. Sulfur-35 (S-35), is incorporated into the nucleic acid backbone by using radiolabeled nucleotides during synthesis.

How are biotin and digoxigenin used as molecular tags?

Avidin is bound to biotin, conjugated to an enzyme which cleaves phosphate groups, allowing detection with substrates.

What is denaturation of DNA?

The breaking apart of a double strand of DNA into two single strands.

What is melting temperature?

The temperature at which the two strands of a DNA molecule are half unpaired.

What is annealing?

The re-pairing of separated single strands of DNA to form a double helix.