5: Manipulation of Nucleic Acids

Question 1

Cutting and joining DNA

Answer 1

Enzymes that degrade nucleic acids.
Are ribonucleases (RNases) or deoxyribonucleases (DNases). 

Exonuclease / endonuclease

Restriction enzymes: endo-

• Recognition site on DNA is 6-8 bp, inverted repeat.
• Type I: cuts DNA 1000 or more bp away from recognition site.
• Type II: cuts in the middle of the site. Most used.

Blunt ends:
- Ends of dsDNA are fully base paired and have no unpaired ss overhang.

Sticky ends:
- Ends of dsDNA that have unpaired ss overhangs, generated by a staggered cut.

DNA ligase covalently links the phosphate backbone of DNA fragments. Easier to ligate sticky ends than blunt ends.

Question 2

Measuring DNA and RNA concentration with ultraviolet lights

Answer 2

Proportion of UV lights absorbed depends on the amount of DNA or DNA.

Proteins: 280 nm

Can measure relative purity of DNA by the A260/280 ratio.
Pure DNA: 1.8
Pure RNA: 2.0

UV light absorption by nucleic acids is due to energy transitions of the delocalized electrons of the aromatic rings of the bases.

Question 3

Labelling of nucleic acids

Answer 3

Incorporating radioisotopes into the DNA.
32P and 35S important in molecular biology.

Fluorescence:
a molecule absorbs light of one wavelength and then emits light of another, longer, lower energy wavelength.

Chemical tagging with biotin:
Vitamin B.
Linked to uracil -> incorporated into DNA. Can bind avidin to biotin and add the Ab’s of the former. The Ab’s can be visualized.

Question 4

Hybridization of DNA and RNA

Answer 4

Sequences that don’t match perfectly can base pair from different DNA double helixes if dsDNA is heated -> ss -> cooling -> DNA hybrid

Question 5

Southern blotting

Answer 5

Method to detect ssDNA that has been transferred to a solid support by using a probe that binds DNA.

• Target DNA is cut into smaller fragments and run on an agarose gel.
• Denaturation -> ss
• Addition of radioactive probe -> forms hybrid with DNA with related seq
• Fragments can bi visualized

Question 6

Northern blotting

Answer 6

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

Mixture of RNA is run on a gel and transferred to a membrane. The membrane is probed as with Southern blotting.

Question 7

Western blotting

Answer 7

No nucleic acid hybridization.

Proteins separated on a gel, transferred to a membrane, and detected by antibodies.

More described in chapter 15 - Transcriptomics

Question 8

FISH - fluorescence in Situ Hybridization

Answer 8

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

Detects the presence of a gene, or corresponding mRNA, within the cell itself.