Nucleic Acids

Question 1

What is a nucleoside?

Answer 1

A purine or pyrimidine base, bonded to a sugar molecule by a glycosidic bond.

Question 2

What is a purine, and name the purine bases.

Answer 2

A double ringed base.

Adenine and Guanine.

Question 3

What is a pyrimidine and name the pyrimidine bases.

Answer 3

A single ringed base.

Cytosine, Thymine and Uracil.

Question 4

Compare the labelling of the carbon atoms in the base vs the sugar molecule.

Answer 4

Base- carbon atoms are numbered from 1-.

Sugar- carbon atoms are numbered with prime.

Question 5

What is a nucleotide?

Answer 5

Compounds consisting of a nucleoside linked to a phosphate group (s).
These form the basic structural units of nucleic acids DNA and RNA.

Question 6

What are nucleic acids?

Answer 6

Polymers of nucleotides.

Molecules that allow organisms to transfer genetic information between generations.

Question 7

Compare DNA to RNA.

Answer 7

DNA-

• Deoxyribose sugars.
• No uracil base.
• Double stranded.
• Storage form of the genetic information.
• Heteropolymer of 4 different nucleotides (ATCG).

RNA- 
-Oxyribose sugars. 
-Uracil bases instead of Thymine.  
-The working copy of the gene.
-Single stranded. 
Heteropolymer of 4 different nucleotides (AUCG).

Question 8

What is a glycosidic bond?

Answer 8

It is the bond formed between the sugar molecule and the base. Water molecule is released.

Question 9

Between which atoms is the glycosidic bond formed?

Answer 9

1’ of the carbon of the sugar molecule, and the N-1 (pyrimidines), or N-9 (purines).

Question 10

What enzyme is involved in nucleotide polymerisation?

Answer 10

DNA polymerase.

Question 11

Outline the process of nucleotide polymerisation.

Answer 11

DNA polymerase, forms hydrogen bonds and covalent bonds in the 5’ to 3’ direction through the polymerisation of the monomer dNTPs.

Question 12

Where are the hydrogen bonds formed in DNA?

Answer 12

Between the complementary bases of the two strands.

Question 13

How is the geometry of the DNA helix maintained?

Answer 13

Through the hydrogen bonds.
They are formed between a purine and pyrimidine.
GC- 3x H-bonds.
AT- 2x H- bonds.

Know structure of bonding!

Question 14

Where are covalent bonds formed in the DNA molecule?

Answer 14

Between the sugar and phosphate of adjacent bases, to form the phosphodiester backbone… 5’ phosphate, and 3’ OH.

Question 15

How was the 3D structure of DNA discovered?

Answer 15

By x-ray diffraction photographs in 1953 by Watson and Crick.

Question 16

What is Chargaff’s rule?

Answer 16

The CG, AT base pairing rules… to maximise the number of effective hydrogen bonds that can form between bases, allowing formation of the most stable conformation.
Pyrimidine always paired with a purine to keep the same distance between the two strands so that the bases can bond, maintaining the geometry.

Question 17

What is the importance of minor and major groves?

Answer 17

I.e. where the backbone is closer/ further apart.
Major grove… more exposed… allows certain proteins to bind to DNA to alter its structure or to regulate transcription or replication.

Question 18

What are the three DNA helix geometries?

Answer 18

A-DNA.
B-DNA.
Z-DNA.

Question 19

What is the most predominant type of structural conformation of DNA in the cells?

Answer 19

B-DNA, occurring under normal physiological conditions.

Question 20

Outline how DNA is packaged.

Answer 20

• A double stranded DNA helix is complexed with histones to form nucleosomes.
• Each nucleosome consists of 8 histone proteins around which the DNA wraps 1.65 times.
• A chromatosome consists of a nucleosome plus the H1 histone.
• The nucleosome folds up to produce a 30n m fibre, forming loops around 300nm in length, which are compressed and folded to produce a 250nm-wide fibre.
• THe tight coiling of the 250nm fibre produces the chromatid of a chromosome.

Question 21

Compare DNA packaging in eukaryotes vs prokaryotes.

Answer 21

Eukaryotes-

• One long, linear dsDNA molecule.
• Bound to a complex mixture of proteins to form chromatin.
• Closed, circular ds DNA molecules in mitochondria and in chloroplast.

Prokaryotes-

• Single, circular, dsDNA molecule.
• Can take the supercoiled, circular, or nicked form, although can also undergo positive and negative supercoiling.
• Generally prokaryotic chromosome is associated with non-histone that help bacteria to compact the DNA to form a nucleoid.
• Extrachromosomal DNA plasmid.

Question 22

What is a nuclease?

Answer 22

Enzyme that can cleave the phosphodiester bond between the monomers of nucleic acids, at specific site.

Question 23

What is an exonuclease?

Answer 23

Works by cleaving nucleotides one at a time from the end of the polynucleotide.

Question 24

What is an endonuclease?

Answer 24

Enzyme that cleave the phosphodiester bond in the middle of the polypeptide chain.

Question 25

What is a restriction endonuclease and name 3 different ones.

Answer 25

An enzyme that cleaves both strands of dsDNA at a specific sequence... staggered cuts. Palindromic recognition site. EcoR1, Pst1, Hpa1.

Question 26

What is a palindromic sequence?

Answer 26

A sequence that reads the same forwards as backwards.

Question 27

List some characteristics of the EcoR1 enzyme.

Answer 27

- Recognises the palindromic sequence GAATTC. | - Forms sticky ends with a 5' overhang.

Question 28

List some characteristics of the Pst1 enzyme.

Answer 28

Recognises specific palindromic sequence CTGCAG. | - Forms 3' overhangs.

Question 29

List characteristics of the Hpa1 enzyme.

Answer 29

- REcognises specific palindromic sequence GTTAAC. | - Cuts betten AT in the middle, forming a blunt end cut.

Question 30

What is alkaline phosphatase?

Answer 30

An enzyme with the physiological role of dephosphorylating compounds.

Question 31

What is alkaline phosphatase used for in the lab?

Answer 31

To remove phosphate monoesters, to prevent self-ligation... undesirable in plasmid DNA cloning.

Question 32

What are DNA ligases?

Answer 32

Enzymes that join DNA, and will catalyse the formation of a phosphodiester link. Sticky end ligation, bringing complementary strands together.

Question 33

Why is blunt end ligation less efficient ?

Answer 33

As you are just bringing two pieces of DNA together i.e. can't match up as efficiently as DNA with sticky ends, so need more DNA to be put in the tube, but can be useful when the sticky ends can't be used.

Question 34

How can DNA strands separate?

Answer 34

DNA denaturation, enzymes, in the lab through heat and high pH.

Question 35

What is the Tm of DNA?

Answer 35

The melting temperature i.e. the midpoint... temperature at which the strands separate, i.e. can overcome the hydrogen bonds.

Question 36

What factor affects the DNA's Tm?

Answer 36

The sequence composition of DNA, i.e. CG has three hydrogen bonds so stronger, need more heat to separate, so higher Tm.

Question 37

What is hyperchromicity?

Answer 37

A material's increasing ability to absorb light. I.e. when DNA goes from an ordered to a disordered structure, the absorbance (A) at 260nm increases. I.e. when DNA is heated... denatured. The increase of absorbance (optical density) of a material.

Question 38

What is hybridisation?

Answer 38

Complementary base pairings between DNA fragments, can form in different combinations.

Question 39

What is the Southern Blot?

Answer 39

Once DNA digestion is run on agarose gel and visualised under UV light, transferred DNA from gel onto membrane, then add ssDNA gene probe that is complementary to the gene you are looking for... cloned fragment. Binds to the complementary strand, so can develop autoradiography... black line where probe has bound to complementary DNA.

Question 40

What is DNA topology?

Answer 40

The coiling of a molecule of DNA.

Question 41

How are different plasmid topoisomers separated by gel electrophoresis?

Answer 41

In order of distance travelled (low- high)... Relaxed/ open circle, linear, supercoiled.