Genetics

Question 1

DNA structure

Answer 1

double helix - double stranded 
anti parallel configuration 3’ 5’
A-T 2 hydrogen bonds
C-G 3 hydrogen bonds   
hydrophillic phosphate deoxyribose backbone

Question 2

Helicase / Topoisomerase

Answer 2

unzip and unwind DNA at replication forks

Question 3

origins of replications

Answer 3

often in AT regions (only 2 hydrogen bonds) for Eukaryotes there are multiple as they have lots of DNA but prokaryotes only have a single OR.

Question 4

Primase

Answer 4

is an enzyme that synthesizes short RNA sequences called primers. These primers serve as a starting point for DNA synthesis. error prone

Question 5

DNA polymerase III

Answer 5

DNA polymerase 3 is essential for the replication of the leading and the lagging strands.fast 5’-3’ polymerase, 3’-5’ exonuclease activity. can proof read too

Question 6

DNA polymerase I

Answer 6

DNA polymerase 1 is essential for removing of the RNA primers from the fragments and replacing it with the required nucleotides. slow 5’-3’ polymerase, 3’-5’ and 5’-3’ exonuclease activity

Question 7

DNA Ligase

Answer 7

joins two DNA fragments by forming new phosphodiester bonds. It is used in cells to join together the Okazaki fragments which are formed on the lagging strand during DNA replication.

Question 8

DNA replication steps

Answer 8

Step 1: DNA unzipping (DNA topoisomerase, helicase)
Step 2: RNA primer synthesis (DNA primase)
Step 3: base pairing (DNA polymerase)
Step 4: phosphodiester bonding between nucleotides
(DNA ligase)

Question 9

Transcription

Answer 9

Synthesis of mRNA by RNA polymerase

Question 10

EUKARYOTES vs PROKARYOTES

Answer 10

DNA in the nucleus transcription occurs inside nucleus then exported in cytoplasm for translation

everything occurs in cytoplasm

Question 11

Constitutive promoter

Answer 11

e.g. tRNA,s, rRNAs, ribosomal proteins,

RNA polymerase

Question 12

Inducible promoters

Answer 12

Promotors subject to up/down regulation (Lac, Pho

operons)

Question 13

Transcription (σ) factors

Answer 13

• protein that binds to promoter region, thereby initiating transcription

Question 14

Promoter

Answer 14

specific non-coding DNA sequences to which RNA polymerase holoenzyme binds

Question 15

Enhancer / silencer

Answer 15

regions of DNA (distal) that bind trans- acting factors to enhance/repress transcription

Question 16

Operator

Answer 16

segment of DNA to which a repressor binds (thereby blocking RNApol binding)

Question 17

Regulon

Answer 17

a group of genes regulated by the same regulatory molecule; may be located non-contiguously on the genome

Question 18

Translation

Answer 18

Step 1: an amino-acyl tRNA binds to a vacant A-site, a spent tRNA dissociates from the E-site

Step 2: a new peptide bond is formed

Step 3: the large ribosome subunit translocates, leaving the 2 tRNAs in hybrid sites

Step 4: the small ribosome subunit translocates,
carrying its mRNAs a distance of 3 nucleotides through the ribosome. The ribosome is “reset” for a next cycle.

Question 19

TRANSFER RNA (tRNA)

Answer 19

tRNAs are covalently bound to amino-acids
via aminoacyl-tRNA synthetase
•tRNA match amino-acids to codons in mRNA

Question 20

Recombinant DNA cloning

Answer 20

Step 1: isolate genomic DNA from organism
Step 2: Amplify target DNA via PCR (and purify) Step 3: Cleave target and plasmid DNA with restriction endonucleases to create ‘sticky ends’ Step 4: Ligate DNA fragments into a cloning vector = create a recombinant plasmid
Step 5: Transform recombinant plasmid into host system that will replicate

Question 21

PCR- Polymerase Chain Reaction

Answer 21

Molecular method to exponentially amplify DNA using a thermostable DNA polymerase and thermal cycles

Question 22

PCR cycle

Answer 22

1. DENATURATION
   (melting) 1 min at 95oC
2. PRIMER ANNEALING
   2 to 60s at 56-64 °C
3. EXTENSION
   4 to 120s at 72 °C

Question 23

Quantitative PCR

Answer 23

In qPCR an fluorescent dye (SYBR) or probe (Taqman) is used to determine the number of amplicons after each PCR cycle. Principle: use of a fluorescent “reporter” bound to a “quencher”

Question 24

Plasmids

Answer 24

circular episomal (extra-chromosomal) dsDNA molecules

Question 25

Transformation

Answer 25

The uptake and incorporation of exogenous genetic material through the cell membrane. For transformation to occur, recipient cells must be in a state of competence.

Question 26

Transduction

Answer 26

is the process by which DNA is transferred from one bacterium to another via bacteriophage (virus). Different methods to perform DNA uptake; the most common:
chemoporation (heat shock), electroporation (electric pulses), conjugation, transduction.

Question 27

Bacterial Conjugation

Answer 27

Exchange of genetic material between bacteria. Requires physical contact (DNA exchanged via Pilli)

Question 28

screening vs selection

Answer 28

selection - defined the ones which have the plasmid (determined from colour blue = insert not present, white = insert present)
Screening - find the ones with the genes of interest

Question 29

Plasmid instability occurs as a result of:

Answer 29

1. DNA mutation (inactivation)
2. Defective plasmid segregation (via low copy number)
3. Insufficient selective pressure (e.g. no antibiotics)

Question 30

Restricition endonucleases

Answer 30

Restriction enzyme, also called restriction endonuclease, is a protein produced by bacteria that cleaves DNA at specific sites along the molecule. Cleave target and plasmid DNA with restriction endonucleases to create ‘sticky ends’

Question 31

Plasmid

Answer 31

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria

Question 32

RNA polymerase

Answer 32

RNA polymerase uses one of the DNA strands (the template strand) as a template to make a new, complementary RNA molecule