MFD5

Question 1

What is recombinant DNA technology?

Answer 1

Genetic engineering and cloning

Question 2

E.Coli K12 Genome

1) What is the shape of its chromosome?
2) a) How many base pairs?
b) How many proteins does that code for?
3) What percentage of genes are of unknown function?
4) The average protein has ___a___ amino acids.

Answer 2

1) circular
2) a) 4.6 million base pairs.
b) 4,400
3) 40%
4) 300

Question 3

What are operons?

2) Name a genome where this occurs:

Answer 3

1) the clustering of genes that encode enzymes of a single biochemical pathway.
2) E.Coli K12 Genome

Question 4

1) What are they?
2) What sort of information do they carry?
e. g.
3) What releases plasmids?
4) What absorbs plasmids?

Answer 4

1) Small circular DNA
2) not essential to survive but are useful
e. g. e.g. genes which make bacteria resistant to antibiotics
3) are released by dead bacteria
4) absorbed by those still living thus genetic information is exchanged

Question 5

Horizontal Gene Transfer in bacteria

Occurs naturally by :

Answer 5

1. Transformation
2. Transduction
3. Conjugation

Question 6

Define

horizontal gene transfer via Transformation:

Answer 6

the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane

Question 7

Define

horizontal gene transfer via Transduction:

Answer 7

injection of foreign DNA by a bacteriophage virus into the host bacterium

Question 8

Define

horizontal gene transfer via • Conjugation:

Answer 8

transfer of genetic material between two bacterial cells in direct contact

Question 9

Is the following transformation, transduction, pilus-mediated transfer, conjugation:
DNA uptake

Answer 9

transformation

Question 10

Is the following transformation, transduction, pilus-mediated transfer, conjugation:
Phage-mediated DNA transfer

Answer 10

transduction

Question 11

Is the following transformation, transduction, pilus-mediated transfer, conjugation:
Mating

Answer 11

pilus-mediated transfer; conjugation

Question 12

DNA is incorporated into the chromosome of the recipient cell via ____a____ after horizontal gene transfer has occured.

Answer 12

homologous recombination

Question 13

What 3 thing ford biotechnology produce:

Answer 13

Production of recombinant proteins e.g. insulin
Develop vaccines e.g. hepatitis B
Generate improved probiotic bacteria

Question 14

What is recombinant DNA technology?

3

Answer 14

1. Creation of recombinant DNA
   - Construction of new combinations of unrelated genes in the test tube
2. Cloning of recombinant DNA:
   –Amplifying the new DNA many times by introducing it into living cells- produces many identical copies (clones).
3. Using recombinant DNA, example:
   -Expressing a cloned gene to produce a protein

Question 15

Why do molecular biologists have this in there toolbox:

enzymes

Answer 15

To manipulate the DNA/RNA

Question 16

Why do molecular biologists have this in there toolbox:

Vectors

Answer 16

Act as a vehicle to carry recombinant DNA into host cell

Question 17

Why do molecular biologists have this in there toolbox:

Taq polymerase

Answer 17

the raw material- usually purified from tissue/cell culture

Question 18

Why do molecular biologists have this in there toolbox:

Reverse transciptase

Answer 18

To amplify –and sometimes express- the recombinant DNA

Question 19

What are restriction enzymes?

+e.g.

Answer 19

1) molecular scissors

e. g. Restriction endonucleases:

Question 20

1) How many base pair palindromic sequences do restriction enzymes recognise?
2) What is a palindromic sequence?

Answer 20

1) 4-8
2) sequence made up of nucleic acids within double helix of DNA and/or RNA that is the same when read from 5’ to 3’ on one strand and 5’ to 3’ on the other, complementary, strand

Question 21

Restriction endonuclease

1) What do they do?
2) What is it naturally produced by and why?

Answer 21

1) Cleave DNA, usually at very specific sequences

2) Naturally produced by bacteria: self-defence mechanism –cleaves foreign (bacteriaphage) DNA

Question 22

What are the 2 types of cleavage, and what ends of they produce?

Answer 22

1. Symmetrical cleavage = blunt ends

2. Asymmetrical cleavage = sticky ends

Question 23

What type of ends are easier to anneal together?

2) What enzymes is used to anneal them?

Answer 23

1) Sticky ends are easier to anneal together with

2) ligase.

Question 24

Give the 5 features of a vector:

Answer 24

• Contain unique restriction sites for insertion of the new DNA.
• Can be introduced into host cell relatively easily
• An efficient origin of replication
• Contain a gene to allow selection of cells which contain the plasmid- eg an antibiotic resistance gene
• May also contain regulatory sequences to allow expression of the inserted gene. (polylinker)

Question 25

What are commonly used as vectors?

Answer 25

plasmids

Question 26

Plasmids

1) What size do they come in?
2) Do they replicate at the same time as the bacterial chromosome?
3) What is their function?

Answer 26

1) 2-200kbp
2) no, replication is independent of bacterial chromosome
3) many functions e.g. self-defence- may carry genes for antibiotic resistance

Question 27

What is a polylinker?

Answer 27

multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids

Question 28

A) Other than plasmids what else can be used as vectors: (2)

B) Why may they be more useful?

Answer 28

A)1)Bacteriophage

2) Cosmids/ phagemids:
b) for cloning larger fragments of DNA

Question 29

What are bacteriophaes?

Answer 29

(viruses which infect bacteria)

•

Question 30

What are Cosmids/ phagemids

Answer 30

genetically engineered hybrids which replicate as a plasmid but can be packaged as a bacteriophage.

Question 31

How would we set about cloning a human gene in E.coli?

Answer 31

1. Create Vector: Cut DNA molecule using restriction enzymes or restriction endonucleases, and then pasting the DNA of interest into the plasmid at the sticky ends using an enzyme called ligase
2. Recombinant plasmid inserted into E.coli host .
3. E.coli cells containing plasmid are selectively cultured
4. Recombinant plasmid is purified or expressed

Question 32

How is bacteria containing pUC18 plasmids selectively cultured?

Answer 32

By growing on agar + ampicillin.

Question 33

How is recombinant plasmid inserted into E.coli host ?

Answer 33

host cell in transformed by CaCl2 on ice +heat shock (42degrees, 2mins) OR electroporation

Question 34

What are the issues with using animal hormones to replace our lack of hormones?

Answer 34

1) Animal protein not identical to human= side effects
2) Difficult to purify
3) Possible contamination with pathogens eg BSE

Question 35

Where is insulin made?

2) What is its function?
3) What disease does it cause?
4) How were symptoms alleviated?

Answer 35

1) Insulin made in the beta-cells of Islets of Langerhans in pancreas
2) Vital for controlling glucose levels in blood
3) Insulin defiency results in diabetes mellitus – death if untreated afflicts millions of people worldwide
4) Alleviate symptoms by regular injections of animal insulin e.g. pigs/ cows

Question 36

1) What is the issue when producing a vector containing the gene for human proinsulin?
2) What is the solution?

Answer 36

1) the human proinsulin gene has 2 introns (non-coding segments)
2) using mRNA for human insulin, it is reverse transcribed proinsulin cDNA (no introns)

Question 37

How is recombinant DNA technology used to improve oral health?

Answer 37

• Recombinant DNA can be used to manipulate genes to produces GM organisms.
• Creation of a genetically altered strain fo Streptococcus mutans, called SMaRT, which does not produce lactic acid.
• The SMaRT replacement therapy technology: protection against tooth decay!