HC1 basics

Question 1

Central Dogma

Answer 1

DNA, RNA, protein, metabolite, phenotype

Question 2

DNA strands synthesised direction

Answer 2

5’- 3’

Question 3

definition DNA synthesis

Answer 3

process of the formation of phosphodiester bonds while hydrolysing the matching dNTP molecule. Energy for the formation of the covalent phosphodiester bond comes from substrate itself.

Question 4

Proofreading

Answer 4

by DNA polymerases, if a nulceotide does not fit, the polymerase recognises it and removes it.

Question 5

Error in newly made strand during replication: remove the mistake how?

Answer 5

scanning proteins MutS and MutL scan DNA for mistakes. One of the strands is partially removed and is newly synthesised

Question 6

Depurination

Answer 6

Nucleotide (G/A) dissapears in DNA. No more base. During replication: one of the strand has a deletion (A-T or C-G misses). Other strand remains normal.

Question 7

Deamination

Answer 7

Loss of amine group. Cytosine looks like a uracil. one of the strands after replication is unchanged, the other will have an A instead of G.

Question 8

dideoxy DNA sequencing (Sanger sequencing)

Answer 8

dideoxynucleotides (ddNTPs) can be incorporated. These are chain terminators and they lack the 3’OH necessary for strand extension. you need ss DNA. The primer used determines the start of the reaction, randomly ddNTPs will be incorporated so you get different size of fragments. Fragments can be separated using gel electrophoresis.

high concentration of dNTPs, low of ddNTPs.

Question 9

shotgun sequencing

Answer 9

whole genome sequencing: genomic DNA is cut up into smaller fragments –> BAC library –> DNA fragments are amplified and inserted into BACs (plasmids) –> sequencing. Then compare overlapping fragments.

Question 10

pyrosequencing + 4 enzymes used

Answer 10

done in small wells. in wells a nanoparticle with DNA on it. When a dNTP molecule is incorporated it supplies PPi and this is converted to ATP. ATP is consumed by luciferase and results in a light signal.
dNTPs need to be added individually, otherwise you cant determine which dNTP was incorporated.

4 enzymes used in pyrosequencing:
1. DNA polymerase
2, sulfurylase: converts PPi into ATP
3. luciferase: uses ATP to produce light
4. Apyrase: degrades unincorporated dNTPs, so a new reaction can start with a new dNTP.

Question 11

Chip: pH change

Answer 11

when a nucleotide is incorporated: release of proton –> results in pH change and can be measured on a chip.

Question 12

PCR: step by step

Answer 12

1. choose primers in desired area.
2. separate DNA strands (95oC)
3. anneal primers (50-55oC)
4. DNA synthesis (72oC)
5. second cycle: sepate DNA strands and anneal primers again, it repeats itself.

Question 13

advantages and disadvantages PCR

Answer 13

+ sensitive and fast
is able to detect VNTR and SNP
- limited to certain size and it needs homologous DNA sequences

Question 14

RT-PCR

Answer 14

1. isolate mRNA
2. add primer (poly T RNA primer), reverse transcriptase
3. cDNA is formed
4. separate strands: eventually get ds cDNA
5. add second primer
6. PCR amplification.

Question 15

advantages and disadvantages RT-PCR

Answer 15

+ sensitive and fast, cDNA does not contain introns
- 5’end RACE for full length of gene families.

Question 16

VNTR

Answer 16

variable number tandem repeats. they generate a fingerprint of an individual. Used to determine allele frequencies

Question 17

SNP

Answer 17

single nucleotide polymorphism. not mutations!

Question 18

DNA replication

Answer 18

1. Helicase unwinds ds DNA by walking over 1 strand 5’-3’ (lagging strand). (leading strand is opened from 3’- 5’ and will be replicated continuously.)
2. DNA polymerase will replicate DNA

Question 19

okazaki fragments

Answer 19

DNA from lagging strand can only be replicated starting from the replication fork: so there are different fragments. for each of these fragments a RNA primer is needed. Fragments are connected together via ligase.