Recombinant DNA techniques

Question 1

what DNA need to Fragmented?

Answer 1

DNA is enormously long and must be fragmented into
manageable sized pieces for any analysis involving a study of sequence.
•Shearing forces produce only random fragmentation.

Question 2

Restriction enzymes

Answer 2

Enzymes purified from bacteria which can cut both
strands of DNA at specific sites
These enzymes cut DNA in a very precise way and only at sites with specific base sequences, hence generating a totally reproducible set of fragments.

Question 3

Why do bacteria make Restriction enzymes?

Answer 3

– Protect bacteria from viruses by degrading incoming viral DNA

Question 4

Several types of restriction endonucleases

Answer 4

– type II: cleavage site is within or close to the recognition site.
• Different restriction enzymes have different sequence specificities

Question 5

Restriction Endonuclease EcoR1

Answer 5

Role in bacteria - to degrade (restrict) foreign DNA

Question 6

Orthodox Type II Restriction Enzymes

Answer 6

• recognition sites are sequence dependent, though with varying degrees of stringency
• always cut both strands in the same way
• most sites are 4-8 nucleotides long and palindromic
• require Mg2+ but not ATP
• used singly or in combination
=> cornerstone of recombinant DNA technology

Question 7

How is DNA cut?

Answer 7

• Enzymes cut near or at recognition sequence
• Recognition sites often palindromic
• For HpaI, cleavage leaves blunt ends
• For EcoRI etc., cleavage leaves staggered ends or
‘sticky’ ends

Question 8

Major Uses of Endonucleases

Answer 8

• Generation of reproducible, manageable-sized family of fragments
– DNA sequencing
• Gene cloning
– recombinant protein expression (e.g. insulin)
• DNA profiling / fingerprinting
– comparative studies, forensics
• Gene mapping
– follow the creation / destruction of sites due to mutation of the gene
sequence
• Gene disruption
– Genome engineering using the Crisp-Cas9, Transcription activator-like
effector nuclease (TALEN) and Zinc finger nucleases

Question 9

How restriction endonuclease is used in Cloning DNA fragments?

Answer 9

Insertion of DNA fragment into bacterial
plasmid
• Plasmid cut open with restriction enzymes
• Insert digested with same restriction enzymes
• Mix plasmid and insert.
Enzymatic ligation:
– DNA ligase

Question 10

Amplification of DNA fragment inserted into a plasmid

Answer 10

Recombinant plasmid DNA introduced into competent
bacteria (i.e. cells that are made transiently permeable to DNA)
• As cells divide, plasmids carrying foreign DNA
multiply
• Selection of transformed bacteria in presence of
antibiotic

Question 11

Typical E. coli expression vector

Answer 11

• Efficient expression:
 Promoter
•Ribosome-binding site
Transcription termination sequence
• Inducible expression:
repressor gene
operator sequence
Selection:
antibiotic
 blue-white selection
(LacZ gene disruption)

Question 12

Polylinker or Multiple

Cloning Site:

Answer 12

Multiple restriction sites to facilitate cloning

Question 13

What is Directional cloning

Answer 13

The use of different restriction sites on each
end of the DNA to clone allows directional cloning
into a plasmid digested with the same enzymes
Polylinker allows you to have some directional cloning
you control where the gene is going
blunt end both side –> is not going to be directional
becuse you can go one way or the othe

Question 14

How can engineered genes can be inserted into host cells?

mammalian cell, plant cell and bacteria

Answer 14

– into mammalian cells via transfection, microinjection
or a virus
– into plants via Agrobacterium tumefaciens or particle bombardment
– into bacteria via electroporation, heat-shock
or chemical transformation

Question 15

Recombinant protein expression

Answer 15

Coding sequence inserted in an expression vector
• Promoter
• Optional: Tag for purification
• Correct orientation
• Cellular host may be important
(E. coli, yeast, insect cells,
mammalian cells)
• Yield
• Folding
• Activity

Question 16

How can site directed mutagenesis be achived?

Answer 16

Site-directed mutagenesis allows to alter specific sequences
– PCR with modified oligonucleotides
– Results in a change in the protein sequence (mutation, deletion or insertion)
• Template DNA is degraded by using methylation-dependent restriction endonuclease (e.g. Dpn I)
the manipulated DNA that you want to amplify is not going to degrade as it is methylated

Question 17

Gene replacement, knockout and

addition

Answer 17

(A) Normal gene completely replaced – providing
information on activity of mutant gene without
interference from normal gene
(B) Normal gene inactivated completely – to see effect of losing the normal gene
(C) Mutant gene added – useful when mutant gene is
dominant over normal gene

Question 18

What is Conditional mutations?

Answer 18

• Use of regulatory DNA sequences allow the selective
regulation of gene expression.
• Bacterial DNA binding domain fused to an eukaryotic activator of transcription.
• Inhibition of the DNA-binding domain by a small molecule leads to gene inactivation.
• Tissue-specific gene activation if the activator fusion protein is only expressed in certain tissues.

Question 19

How do bacteria protect their own DNA from being cut by restriction enzyme?

Answer 19

Bacteria protect their DNA by methylating the
Nucleotides to prevent the restriction enzyme
binding and degradation of the DNA
done by enzyme Methylase (A)

Question 20

How directional cloning is done?

Answer 20

Directional cloning is when two different restriction enzymes is used to cut the DNA insert and the
vector (plasmid) to produce different sticky end at the end each DNA insert and the plasmid which
are compatible. For example, HinIII enzyme is used to cut one end of the DNA and one end of the
plasmid and another enzyme such as BamHI is used to cut the other end of DNA and the plasmid. This
is done to make sure that the DNA is ligated in correct orientation in the vector and also avoid the
vector self-ligation