Chapter 5: Manipulation of Nucleic Acids

• Define:

  • Exonuclease: attack the ends of molecules

  • Endonuclease: cleave the nucleic acid chain in the middle

  • Isoschizomer: Two restriction enzymes 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 only 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: 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 the production of restriction fragments of different lengths.

  • Autoradiography: Laying a piece of photographic film on top of a gel or membrane to identify the exact location of the radioactive DNA.

  • Scintillation counting: 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 by using a probe that binds DNA.

  • Northern blotting: Hybridization technique in which a DNA probe binds to an RNA target molecule.

  • Western blotting: 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.

• Describe why restriction enzymes evolved and how they function in nature

  • They evolved as a defense mechanism from foreign DNA (such as bacteriophages) by recognizing and cleaving specific DNA sequences. They are basically like a bacterial immune system. 

• Describe the naming system for restriction enzymes

  • Identifies the bacterial species (strain) that the enzyme was purified from

  • First letter (capitalized) from Genus, next letters (lower case) from species

  • Sometimes the strain of the bacterium is represented

  • Roman numeral indicates the number of restriction enzymes found in the same species

    • EX.) Hpa II (Haemophilus parainfluenzae)

• Describe the two major classes of restriction enzymes

  • Type I: Specific recognition site, methylates the recognition site, cuts the DNA a thousand or more base pairs away from the recognition site. Very unpredictable

  • Type II:  Specific recognition site, which Cuts the DNA within the recognition site. The exact position of the cut is unknown; very useful for molecular biology, the cut generates either blunt ends (cuts both strands of DNA at the same point) or sticky ends (staggered cut)

• Describe how spectrophotometer (NanoDrop) can be used to determine DNA concentration in a solution

  • measuring the amount of ultraviolet light absorbed by the DNA sample at a specific wavelength (260nm), which is directly proportional to the concentration of DNA present

• Interpret spectrophotometer results: A260/280 and A230/260 readings

  • A260/280: “pure” DNA, minimal protein contamination

  • A230/260: minimal presence of residual chemicals like phenol or salts

• Describe phosphoramidite method of DNA synthesis

  • Method for artificial synthesis of DNA that utilizes the reactive phosphoramidite group to make linkages between nucleotides

  • 3’ to 5’ synthesis

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

  • Phosphoramidite nucleotides (single phosphorus) - activates

• Name the two radioactive isotopes most often used to label nucleic acid and how they are incorporated and detected

  • 1. phosphorus-32 (P-32)

  • 2. sulfur-35 (S-35)

  • incorporated into the nucleic acid backbone by using radiolabeled nucleotides during synthesis

  • Detected by scintillation counter or autoradiography

• Name two molecular tags used to label DNA and how they are detected

  • 1. Biotin

  • 2. Digoxigenin

  • First, avidin is bound to the biotin. The avidin is conjugated to an enzyme called alkaline phosphatase, which cleaves phosphate groups from various substrates. Second, a substrate such as X-phos (shown) or lumi-phos (not shown) is added

• Define:

  • Denaturation of DNA: the breaking apart of a double strand of DNA into two single strands

  • Melting temperature: The temperature at which the two strands of a DNA molecule are half unpaired

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

• Define:

  • Autoradiography: Laying a piece of photographic film on top of a gel or membrane to identify the exact location of the radioactive DNA.

  • Scintillation counting: 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 by using a probe that binds DNA.

  • Northern blotting: Hybridization technique in which a DNA probe binds to an RNA target molecule.

  • Western blotting: 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.