Nucleic acids part 4

Question 1

What enzymes are used to cleave DNA and why must DNA be cleaved to be sequenced?

Answer 1

DNA molecules are too large to be sequenced intact - so they must be cleaved.

Restriction endonucleases are used to cleave DNA to give DNA fragments.

Question 2

What is the Sanger method used for and what is the DNA fragment incubated with?

Answer 2

The Sanger method is used by DNA sequencing machines to sequence DNA.

The DNA fragment to be sequenced is incubated at ~70°C with:

• DNA polymerase I
• A DNA primer (Klenow fragment)
• The four DNTP monomers
• A small amount of a dideoxynucleoside triphosphate (ddNTP).

Question 3

What are ddNTPs and what role do they play in the Sanger method?

Answer 3

ddNTPs are missing an -OH group at the 3’ position, meaning no phosphate group can be attached.

ddNTPs can be radiolabeled or labelled by attachment of a fluorescent dye.

Question 4

How does the Sanger method work?

Answer 4

A strand complementary to the restriction fragment grows from the 5’ end of the primer.

In most cases a normal dNTP is incorporated into the growing DNA chain, but occasionally one of the ddNTPs is incorporated instead.

This causes the DNA biosynthesis of the chain to stop.

At the end the product is a mixture of DNA fragments of all the possible lengths, each one terminated by a ddNTP.

After gel electrophoresis of the product mixture, the identity of the terminal ddNTP is obtained for each DNA fragment.

This info allows the sequence of bases of the restriction fragment to be sequenced.

Question 5

What are 2 ways in which the automated Sanger method vary from the original method and what is one way in which it is superior?

Answer 5

The automated Sanger method:
- Requires fluorescent labelling.
- All of the chain-growth reactions are carried out in a single vessel.

The automated Sanger method is greatly accelerated by automation.

Question 6

At what rate can modern systems using the Sanger method sequence base pairs (per day) and how long would it take to sequence the human genome at this rate?

Answer 6

Advanced systems can sequence ~10^6 base-pairs per day.

At this rate it would take ~10 years to sequence the human genome.

Question 7

What happens when water and dioxygen are exposed to UV light or ionising radiation?

Answer 7

Water - generates hydroxyl radicals

dioxygen - generates peroxy radicals

Question 8

What is strand breaking and how is it caused?

Answer 8

The hydroxy and peroxy radicals can react with the deoxyribose of the sugar-phosphate backbone causing the chain to cleave.

Question 9

What is radical oxidation and how is it caused?

Answer 9

The hydroxy and peroxy radicals can modify the nucleotides through oxidation.

Question 10

What is an example of a protein-DNA interaction, how can this interaction be interrupted and what can this lead to?

Answer 10

The interaction of gene transcription factor AP-1 with Z-DNA can be interrupted by DNA modification.

Interruption can lead to cancer.

Question 11

What are photochemical reactions, what causes them and how can they be reversed?

Answer 11

UV light can cause a photochemical reaction in DNA.

This is where two adjacent purines on one strand dimerise.

This results in the misreading of DNA during replication which can cause mutations.

These mutations can be removed by enzymes.

Question 12

What is the alkylation of DNA and how is it caused?

Answer 12

Alkylating agents are chemicals which transfer an alkyl group to a nucleophile.

They react with heteroatoms in the nucleotide bases.

Question 13

What can alkylation at N-7 cause, and what is an example of a chemical that can cause it?

Answer 13

Dimethyl sulphate is a powerful electrophile and can alkylise N-7.

Alkylation at the N-7 position may affect the lactam/ lactim equilibrium of the nucleotide base.

Question 14

What is a local breathing mode and what can occur during them?

Answer 14

During local breathing modes, the hydrogen bonding between base pairs is disrupted.

Hydrogen bond acceptor groups (with lone pairs) are then able to react with electrophiles.

Question 15

What are two types of mustards, what is DNA strand cross-linking and how can mustards be used medicinally?

Answer 15

Sulphur mustards and nitrogen mustards

Mustards react at the N-7 position of purines, but have two electrophilic positions and therefore react with both DNA strands.

This leads to covalent cross-linking of DNA strands which cannot separate.

Mustards are crude anticancer agents.

Question 16

What are biologically activated alkylating agents an example of one and what it does?

Answer 16

Some chemicals are not DNA alkylating agents initially.

However they are modified by the body during excretion generating activated alkylating agents.

Vinyl chloride - converted to 2-chloroethanal in the liver.

2-chloroethanal can cause liver cancer.

Question 17

What are N-nitrosoureas and how are they specific?

Answer 17

N-nitrosoureas - developed as selective cancer agents, unreactive until biological activation.

Solid tumours have a poorly developed blood supply, cannot obtain enough oxygen, become anoxic, leading to a pH ~5.5.

The acidic environment activates the anticancer agent.

Question 18

What are chain terminators?

Answer 18

Chain terminators are modified nucleosides that prevent the continued growth of a nucleic acid chain during replication.

Question 19

What conditions is the chain terminator Acyclovir used to treat and how does it function as treatment for the conditions?

Answer 19

Used to treat Infectious diseases caused by DNA viruses such as herpes and shingles.

Acyclovir is activated by the conversion of the hydroxyl group into a triphosphate.

It is then taken into the viral DNA polymerase forming an enzyme-substrate complex with no C-3’ hydroxyl group to add to the next nucleotide.

Question 20

What condition is the chain terminator AZT used to treat and how does it function as treatment for the condition?