MMG2040 chapter 5 - manipulation of nucleic acids

Define exonuclease, endonuclease, isoschizomer, neoschizomer, star activity,
sticky ends, restriction map, RFLP, autoradiography, scintillation counting,
Southern blotting, Northern blotting, Western blotting, FISH

exonuclease: nucleases that attack ends of molecules

endonuclease: nucleases that cleave middle of nucleic chains

isoschizomer: 2 restriction enzymes that share the same recognition site

neoschizomer: subset of isoschizomer, recognize the same sequence but cleave at different positions

star activity: imprecise random cleavage due to unnatural reaction conditions

sticky ends: cut DNA in a staggered cut, residues can be paired to be bonded ends

restriction map: diagram showing the location of cut sites on DNA for variety of restriction enzymes

RFLP: Difference in restriction sites between 2 related DNA molecules that result in multilength fragments, small DNA changes that can eliminate a restriction site —> analysis of molecular polymorphisms

autoradiography: Laying a piece of photographic film on top of a gel or membrane in order 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 (DNA)

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

Western blotting: Detection technique in which a probe, usually an antibody, binds to a protein target molecule

FISH: Fluorescence In Situ Hybridization, 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

restriction endonuclease - recognizes a specific sequence within a DNA molecule and make a db strand cute

• recognition sites are 4-8 bases long, usually in palindromes, binds and cuts DNA

• modification - methylation (adding methyl groups) of recognition sites, prevents cut of bacterium’s own genome

  • protect DNA from the corresponding restriction enzymes by adding methyl groups at the recognition site

evolution:

• bacterial immune system - foreign DNA entering a bacterial cell is most likely due to virus infection, and natural defense systems have evolved to combat the infection

• When viruses attack bacteria, the virus protein coat is left outside and only the virus DNA enters the target cell. The viral DNA will take over the victim’s cellular machinery and use it to manufacture more virus particles unless the bacteria fight back

  • used to distinguish between bacterial/invading DNA

  • degrades foreign DNA without endangering bacterial DNA

Describe the naming system for restriction enzymes

• Identifies bacterial species that an enzyme is purified from

  • First letter (capitalized) from genus

  • next letters (lowercase) from species

  • strain can be represented

  • roman numerals indicate # of restriction enzymes found in same species

    • ex. EcoRI, EcoRII

Describe the two major classes of restriction enzymes

Type I

• Specific recognition site that is methylated

• Cuts DNA 1000+ bp from recognition site

• unpredictable, not useful for molecular bio

Type II

• Specific recognition site and cuts DNA within this site

• exact position is known, very useful for molecular bio

• generates either blunt OR sticky ends

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

uses absorbance of UV light to determine concentration, the
amount of UV light absorbed by the unknown DNA is then determined and plotted on the standard curve to deduce the concentration

• The aromatic rings of the bases found in DNA and RNA absorb ultraviolet (UV) light with an absorption maximum at 260 nm

• If a beam of UV light is shone through a solution containing nucleic acids, the proportion of the UV absorbed depends on
  the amount of DNA or RNA.

• UV light is preferentially absorbed by the purine and pyrimidine rings —> the bases are more spread out and absorb more radiation. In a DNA double helix, the bases are stacked on top of each other and relatively less UV light is absorbed

• In single-stranded RNA (or in single-stranded DNA), UV absorption by nucleic acids is due to energy transitions of the delocalized electrons of the aromatic rings of the bases.

• When the bases stack, electrons interact and no longer absorb UV radiation so readily —> shielding effect

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

• DNA/RNA absorb UV rays at 260 nm

• proteins absorb UV at 280

• relative purity: assessing purity of DNA extraction by measuring absorbance at A260/280 (ratio)

  • if everything has gone right it is approx. 1.8 (little protein, DNA isolated)

  • if protein is present, ratio , 1.8

  • if RNA is present, ratio > 1.8

  • pure RNA is approx. 2.0

A230/260 readings - This ratio is used as a secondary measure of nucleic acid purity

• The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values

• Expected 260/230 values are commonly in the range of 2.0-2.2

• If lower than 2.0-2.2, indicates the presence of contaminants which absorb at 230 nm

Describe phosphoramidite method of DNA synthesis

chemical synthesis de novo

• porous glass beads in a column that serves as a solid support

  • single phosphorus phosphoramidite adds single nucleotide at a time

  • nucleotide binds to glass bead

  • DMT group blocks the 5’-OH between nucleotides

  • adding 3’-5’

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

32P or 35S —> detected by scintillation counter to measure amount of radioactivity

• scintillants emit a flash of light when absorbing

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

chemical tagging

• adding biotin to link to uracil (UTP/dUTP)

• digoxigenin - linked to uracil (UTP/dUTP)

• detected by

fluorescent labelling

• excitation of fluorophore causes fluorescence

• adds fluorophores to DNA

• detected by photodetector to measure fluorescence

Define denaturation of DNA, melting temperature, and annealing

denaturation: dna melting to separate the strands via chemical treatment, or most commonly heating up

melting temperature: temperature at which DNA begins to separate from each other

annealing: bringing down temperature for strands to rebond h-bonds and “stick” back together