Recombinant DNA Technology

Question 1

What is the structure of most bacterial chromosomes?

Answer 1

Double-stranded circular DNA

Question 2

Why is circular DNA considered more stable than linear DNA in bacteria?

Answer 2

It has no ends for exonucleases to degrade.

Question 3

What mechanisms help package the negatively charged bacterial DNA?

Answer 3

Supercoiling and DNA-bending proteins

Question 4

Where is the bacterial genome located?

Answer 4

In the cell cytoplasm

Question 5

Where does bacterial chromosome replication typically start?

Answer 5

At the origin of replication (oriC)

Question 6

How does bacterial replication proceed from the origin?

Answer 6

Bi-directionally

Question 7

What happens to bacterial chromosomes during rapid cell division?

Answer 7

Replication often begins again before the cell has finished dividing.

Question 8

What are ORFs and what do they encode?

Answer 8

Open reading frames; encode mRNA which leads to protein production.

Question 9

How are transcription and translation related in bacteria?

Answer 9

They are coupled and occur simultaneously.

Question 10

How is gene expression mainly regulated in bacteria?

Answer 10

At the transcriptional level.

Question 11

How much can bacterial genome size vary?

Answer 11

From < 1Mb to > 10Mb

Question 12

Why do environmental bacteria tend to have larger genomes?

Answer 12

They require more genes to adapt to changing environments.

Question 13

What kind of genes are retained in bacteria with reduced genomes?

Answer 13

Essential housekeeping genes.

Question 14

What are the types of point mutations caused by DNA polymerase errors?

Answer 14

Transitions and transversions.

Question 15

How can recombination affect bacterial genomes?

Answer 15

It can invert or delete bacterial genome segments based on sequence orientation.

Question 16

What factors can increase mutation rate in bacteria?

Answer 16

Mutagens like base analogues, radiation, and oxidative stress.

Question 17

What are the major mechanisms of horizontal gene transfer in bacteria?

Answer 17

Conjugation, transduction, and transformation

Question 18

What role do plasmids play in horizontal gene transfer?

Answer 18

They transfer genes between bacteria, often via conjugation.

Question 19

How can transposable elements assist horizontal gene transfer?

Answer 19

By moving chromosomal genes onto plasmids.

Question 20

What type of DNA are plasmids made of?

Answer 20

Circular double-stranded DNA.

Question 21

What determines whether a plasmid is maintained in a population?

Answer 21

Natural selection and the fitness cost vs benefit to the host.

Question 22

What is a selfish plasmid?

Answer 22

A plasmid that persists by enforcing its own replication, despite a fitness cost.

Question 23

What is an insertion sequence (IS)?

Answer 23

A transposable element that can disrupt genes or activate their expression.

Question 24

What sequences are essential for a transposition?

Answer 24

Inverted Repeats (IRs).

Question 25

What is a composite transposon?

Answer 25

A structure flanked by IS elements that allows movement of genes between chromosome and plasmids.

Question 26

How do complex transposons differ from composite ones?

Answer 26

They include additional genes like resolvase and can copy-paste themselves.

Question 27

What is the function of integrons?

Answer 27

To capture gene cassettes, especially antibiotic resistance genes.

Question 28

What is cloning?

Answer 28

Inserting a gene of interest into a plasmid, introducing it into bacteria, and selecting colonies that contain the desired recombinant DNA.

Question 29

Where do exonucleases cut?

Answer 29

Cleaves the phosphodiester bonds at DNA ends.

Question 30

Where do endonucleases cut?

Answer 30

Cleaves internal phosphodiester bonds, generating DNA fragments.

Question 31

What does S1 Nuclease cut?

Answer 31

ssDNA, bit can nick dsDNA under certain conditions (e.g. long incubation periods).

Question 32

What does DNase I cut?

Answer 32

Both ssDNA and dsDNA non-specifically to produce small fragments.

Question 33

What is a restriction endonuclease?

Answer 33

highly sequence specific enzymes that cut dsDNA at defined sites. Essential for cloning.

Question 34

What is a Type II Restriction Modification Enzyme?

Answer 34

An endonuclease that cuts at defined positions close to/ within its recognition sequence.

Question 35

How many Type II RM enzymes have been discovered and characterised?

Answer 35

>3,500

Question 36

How many different DNA sequences do type II RM enzymes recognise?

Answer 36

~350

Question 37

How many amino acids are Type IIP RM Enzymes?

Answer 37

250-350. Simplest and smallest.

Question 38

What percentage of enzymes used in molecular biology do Type IIP RM enzymes account for?

Answer 38

>90%

Question 39

What does the P in Type IIP RM enzymes stand for?

Answer 39

Palindromic. They recognise palindromic sequences 4-8bp in length.

Question 40

What is an example Type IIP RM enzyme?

Answer 40

EcoRV

Question 41

What sequence does EcoRV recognsie?

Answer 41

5'-GATATC-3'

Question 42

When EcoRV binds DNA, to what degree does it bend it, and why?

Answer 42

50 degrees to bring the phosphate closer to the enzymes active site.

Question 43

What type of ends does EcoRV produce?

Answer 43

blunt ends (cuts both strands directly opposite).

Question 44

What does the S stand for in Type IIS RM enzymes?

Answer 44

'shifted' due to their cleavage site being shifted.

Question 45

What type of sequences do type IIS RM enzymes recognise?

Answer 45

Asymmetric sequences but cleave outside the recognition site.

Question 46

What is an example of a type IIS RM enzyme?

Answer 46

FokI

Question 47

How far away from the binding site does FokI cleave?

Answer 47

9-13bp away

Question 48

FokI is monomeric but...

Answer 48

forms transient homodimers on DNA to cleave both strands.

Question 49

What type of ends does FokI produce?

Answer 49

sticky ends - helpful for ligation and cloning.

Question 50

In FokI, what does the flexibility of the cut site depend on?

Answer 50

linker strength and helical geometry.

Question 51

What type of restriction endonuclease is HindIII?

Answer 51

Type IIP RE

Question 52

What ends does HindIII produce?

Answer 52

5' sticky ends

Question 53

What palindromic sequence does HindIII recognise?

Answer 53

TCGA

Question 54

What is the purpose of agarose gel electrophoresis?

Answer 54

visualise DNA and estimate DNA fragment size.

Question 55

To what pole of the electric field does DNA migrate towards in agarose gel electrophoresis?

Answer 55

positive pole

Question 56

What is considered a low % of agarose and how does it affect the gel?

Answer 56

0.5% - larger pores better for large DNA fragments.

Question 57

Ethidium Bromide is used to visualise DNA on agarose gel, how?

Answer 57

Intercalates DNA, fluoresces under UV, is a mutagen.

Question 58

What is considered a high % of agarose and how does it affect the gel?

Answer 58

2% - smaller pores better for smaller DNA fragments.

Question 59

What are two ways of sizing DNA fragments on agarose gel?

Answer 59

DNA ladder (e.g. digested lambda DNA) as a reference, or can plot a calibration curve for precise sizing.

Question 60

How does plasmid DNA run on gel electrophoresis?

Answer 60

Unpredictably due to supercoiling, usually faster.

Question 61

How does a nicked/ open circle plasmid run on gel electrophoresis?

Answer 61

Slower due to relaxed conformation.

Question 62

What mathematical equation links migration to molecular mass?

Answer 62

D = a - b (logM) D = distance moved M = molecular mass a and b = constants that depend on electrophoresis conditions.

Question 63

SDS-PAGE. A low % acrylamide creates what size pores?

Answer 63

Large

Question 64

SDS-PAGE. A high % acrylamide creates what size pores?

Answer 64

Small

Question 65

In SDS-PAGE, what stain is used to visualise the proteins?

Answer 65

Coomassie Brilliant Blue

Question 66

What enzyme is typically used in labs to ligate DNA fragments?

Answer 66

T4 DNA Ligase

Question 67

How does T4 DNA ligase join two DNA fragments?

Answer 67

Uses ATP to add AMP to 5' phosphate and forms a phosphodiester bond with 3' hydroxyl.