Lab 6 - Nucleic acids

Question 1

Nucleic acid groups

Answer 1

• Ribonucleic acids (RNA): contains ribose

- Deribonucleic acids(DNA): contains 2-deoxy ribose

Question 2

Determination of the conc. of RNA and DNA extracted from the liver

• The process?
• Materials?

Answer 2

• Separate from free nucleotides and oligonucleotides, by using cold PCA (polynuclotides not soluble). Hydrolyze into bases, pentoses and phosphates in hot PCA. Measue the dissolved pentose.
• Liver from pig, quartz sand, physiological saline, PCA

Question 3

How to estimate nucleic acid

Answer 3

Pentose after hydrolyzing.
Spectophotometry or UV-photometry.
Abs max(clear RNA and DNA)=260nm
Protein contamination=280nm
Abs 260/Abs 280: 1.8-2

Question 4

Determination of RNA by measuring the ribose conc.

• Component used?
• Product?
• Observation?
• Materials?

Answer 4

• Conc. HCl in orcinol reagent forms furfural from the ribose, with deliberation of 3 H2O.
• Furfural reacts with 2 Orcinol and form a condensation prod.
• Green prod.
• Materials:Hot PCA, Orcinol reagent: orcinol+HCl+FeCl3+distilled water, Spectophotometer

Question 5

Determination of DNA conent by measuring the 2-deoxyribose conc.

• Principle?
• Observation?

Answer 5

• In acidic milieu, 2-deoxyribose is converted into beta-hydroxy-levulinaldehyde by loss of water
• Blue complex

Question 6

Isolation of DNA from pig spleen

• Criterias?
• How to separate deoxyribonucleoproteids from other macromolecules?
• How to remove the proteins?

Answer 6

• Must inhibit the DN-ase(deoxyribonuclease) activity, with citratebuffer that binds to Ca2+ and Mg2+(cofactors for DN-ase)
• Deoxyribonucleoproteids are soluble in water and sol. of high salt conc. but insoluble in sol. of low salt conc.
• Removed with chloroform/amylalcohol, and DNA can be ppt. with ethanol.

Question 7

Cleavage of the A-phage DNA by restriction endonucleases:

• What are restriction endonucleases?
• What are the sequences that restiction endonucleases can regonize?
• How can the DNA fragments be separated?
• What is the restriction endonucleases studied on?
• How can the DNA be seen?

Answer 7

• Enzymes from bacteria that can recognize specific sequences of deoxyribonucleotides with double symmetri-generally 4-8 base pairs, and cut them.
• Palindrome sequences
• By gel electrophoresis
• BamHI, HinIII and EcoRI
• UV light, if the agarose contains ethidium bromide - pink.

Question 8

What is recombination?

Answer 8

A process when DNA fragments are rearanged by the religation, or inserted into the DNA of a foreign organism. Makes it possible to transfer genes from one organism to another.

Question 9

What are the producing bacteria, recognized sequence and produced sticky end from the restiction endonyckeases?

Answer 9

• BamHI: Bacillus amyloliquefasciens, GGATCC, CTAG
• HindIII: Haemophilus influenzae, AAGCTT, TCGA
• EcoRI: Escherichia coli, GAATTC, TTAA

Question 10

What are bacteriophages

Answer 10

Viruses that can infect bacteria and replicate in them.

Question 11

Which bacteriophage is suitable for preparative work and why?

Answer 11

Gamma-phage: a double stranded, circular DNA of rel. small molecular mass. Contains 5 GAATTC sequences