Recombinant DNA

Question 1

What is gene cloning?

Answer 1

• A fragment of genomic DNA that includes the gene to be cloned is inserted into a vector (circular DNA mol). This is then called a recombinant DNA molecule.
• The vector transports the gene into a host cell (usually bacteria)
• Inside host cell vector multiplies, producing numerous identical copies, not only of itself but also of the gene it carries.
• Host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place
• After lots of cell divisions, a colony (or clone) of identical host cells is produced

Question 2

Why is gene cloning important?

Answer 2

• The technique can provide a pure sample of an individual gene, separated from all the other genes in the cell
• Each recombinant DNA mol has 1 fragment of genomic DNA, usually each recombinant DNA mol is transported into a single host cell, so each colony contains cells with multiple copies of just one gene fragment

Question 3

What is the key to success in gene cloning?

Answer 3

The ability to identify the particular clone of interest from many different clones that are obtained

Question 4

Why must Genomic DNA be cleaved?

Answer 4

1. To isolate a single gene from inside a large DNA mol

2. Large DNAM mols will need to be cut simply to produce fragments small enough to be carried by the vector

Question 5

What are the functions of cloning?

Answer 5

1. Production of large numbers of rDNA (recombinant DNA) molecules from a limited quantity of starting material
   (million-fold increase)
2. Purification of desired recombinant DNA fragment
   a. Achieved because it is very unusual for a cell to take up more than 1 DNA mol during transformation
   b. Each cell produces a single colony

Question 6

What types of DNA are needed in DNA purification?

Answer 6

1. Total Cell DNA: [chromosomal DNA]
   a. Genomic DNA of an organism along with any additional DNA mols, such as plasmids, that are present
   b. Is the source of material from which to obtain genes to be cloned
2. Pure vector DNA: [plasmid or phage]
   a. Preparation of plasmid DNA follows same basic steps as purification of total cell DNA
   b. Only difference is that at some stage the plasmid DNA must be separated from the main bulk of chromosomal DNA also present in the cell.

Question 7

Steps of DNA Purification

Answer 7

1. Preparation of a cell extract
2. Removal of protein contaminant by phenol extraction
3. Concentration of DNA
4. Plasmid purification by alkaline denaturation

Question 8

How is a cell extract prepared for DNA Purification?

Answer 8

1. Cell lysis: Disrupt cell wall and cell membrane
   - With gram-neg bacteria: weakening of cell wall done through EDTA (ethylenediamine tetraacetate) which chelates Mg2+ essential for preserving overall structure of the cell envelope, and also it inhibits cellular enzymes that could degrade DNA.
   - Detergent SDS (Sodium dodecyl sulphate) is used to aid cell lysis by removing lipid molecules and thereby disrupting the cell membranes.
2. Centrifugation to remove cell debris

Question 9

How are protein contaminants removed from cell extracts?

Answer 9

• Phenol added to cell extract
• Centrifugation separates layers
  o Aqueous later on top containing DNA & RNA
  o Organic layer on the bottom (phenol)
  o Interface of coagulated proteins in between the 2 layers.

Question 10

How is DNA concentrated in DNA Purification?

Answer 10

Most commonly ethanol precipitation is used; in the presence of salt and at a temp>=-20 C absolute ethanol efficiently precipitates polymeric nucleic acids

Question 11

What is the role of salt in DNA Concentration?

Answer 11

o Neutralizes charges on sugar phosphate backbone

o Commonly used salt is sodium acetate – breaks up in sodium and acetate in solution

o Na+ neutralizes neg charge on PO3- groups – makes them far less hydrophilic, and less soluble in water.

Question 12

What is the role of ethanol in DNA concentration?

Answer 12

o Electrostatic attraction between Na+ and PO3- ions dictated by Coulomb’s Law, which is affect by dielectric constant of solution.

o Ethanol lower dielectric constant (water has very high constant) making it easier for Na+, PO3- interaction

Question 13

How is Plasmid DNA purified?

Answer 13

By alkaline denaturation

• Narrow pH range at which non-supercoiled DNA is denatured, whereas supercoiled plasmids are not.
• If sodium hydroxide is added to a cell extract so that pH is adjusted to 12-12.5, then H-bonding in non-supercoiled DNA mols is broken, causing the double helix to unwind and chains to separate.
• Acid now added, denatured bacterial DNA strands reaggregate into a tangle mass. Insoluble network can be pelleted by centrifugation, leaving plasmid DNA in the supernatant.

Question 14

What class of restriction enzyme is used in Gene cloning?

Answer 14

Type II

Question 15

What are blunt ends?

Answer 15

Simple double-stranded cut in the middle of the recognition sequence resulting in a blunt or flush end

Question 16

What are sticky ends?

Answer 16

Formed when 2 DNA strands are not cut at exactly the same position

• Usually staggered by 2 or 4 nucleotides.

Question 17

What is the process of Gell electrophoresis?

Answer 17

• DNA is loaded into a well cut out of the gel, then a current is run through the buffer and gel
• DNA separates into bands of different-sized fragments – smallest travels furthest towards the + terminal
• Agarose gel is stained with Ethidium Bromide and irradiated with ultraviolet light which allows us to visualize the DNA bands

Question 18

Name 2 electrophoresis gels and their pore sizes

Answer 18

1. Agarose gell - 200-20kb

2. Polyacrylamide - <200bp

Question 19

What are 3 restriction enzymes that produce blunt ends?

Answer 19

1. Alu I
2. Pvu II
3. Hae III

Question 20

What are 3 restriction enzymes that produce complementary sticky ends?

Answer 20

1. Bam HI
2. Bgl II
3. Sau 3A

Question 21

What is ligation?

Answer 21

The annealing of complementary bases on sticky ends of genomic DNA and vector DNA

Question 22

What ligase is used in gene cloning?

Answer 22

• T4 ligase
• Requires ATP as a co-factor
• Catalyses formation of phosphodiester bonds between adjacent nucleotides

Question 23

How does ligation occur?

Answer 23

• DNA ligase T4 catalyses joining of 3’-OH to the 5’-P
  1. AMP nucleotide (attached to a lysine residue in the enzymes active site) is transferred to 5’-P

2. AMP-phosphate bond is attacked by 3’-OH, forming a covalent bond and releasing AMP
   - To allow the enzyme to carry out further reactions, AMP in active site must be replenished by ATP dephosphorylation.

Question 24

What reaction conditions must be present for ligation?

Answer 24

• High total DNA concentration
  o Increases frequency of intermolecular associations. Higher total DNA conc increases chances of 1 end of DNA coming into contact with vector DNA and not itself
• Ratio of insert DNA kept at 2-3x higher than that of the vector
  o Avoids vector-vector ligation
  o Ensures that each vector acquires insert DNA

Question 25

What is present in the ligation mix?

Answer 25

o Desired recombinant molecule

o Unligated vector molecules

o Unligated DNA fragments

o Vector molecules that have re-circularized without new DNA being inserted (“self-ligated” vector)

o Recirculated genomic DNA

o Recombinant DNA molecules that carry the wrong inserted DNA fragment.

Question 26

What is transformation?

Answer 26

The uptake of plasmid DNA competent by bacterial cells

Question 27

How can a cell be made ‘competent’?

Answer 27

By being suspended in ice-cold 50mM CaCl2

Question 28

Steps of Transformation

Answer 28

1. Early log-phase cells harvested & re-suspended in ice cold 50mM CaCl2
2. Ice 30mins
3. Plasmid DNA added (The ligation mix)
4. Ice for 10mins
5. Heat shock for 2mins at 42 degrees Celsius
   a. Makes lipids in cell membrane much more fluid, allows plasmid to cross the membrane
6. Add cells to nutrient broth; incubate 37 deg C – 30mins
7. Plate cells on selective agar

Question 29

Efficiency of transformation

Answer 29

• Transformation is relatively inefficient
• ±0.01% uptake of available DNA molecules
• Only a small proportion of total number of cells take up DNA (1/10^5)
  o Why its very unlikely to find a cell with more than one plasmid

Question 30

What is insertional inactivation?

Answer 30

When genomic DNA is inserted into a target gene in the vector molecule, inactivating that gene.

Question 31

What is replica plating?

Answer 31

• Velvet on the bottom of a wood block is used to transfer colonies from 1 medium to another in the same spatial positioning.

Question 32

What are the properties of an ideal plasmid vector?

Answer 32

1. Low molecular weight
   a. Plasmid is easier to handle (less sensitive to shearing)
   b. Usually multiple copy (per cell)
   i. Improves isolation
   ii. Gene dosage effects for cloned genes
   c. Less chance of multiple restriction sites for any particular restriction enzyme
2. Ability to confer readily selectable phenotypic traits on host cells (report gene)
   a. Efficiency of transformation is very low – needs to be clear when transformation has happened
   b. Generally use an antibiotic resistant gene
3. Single sites for a large number of restriction enzymes, preferably in genes with scoreable (observable) phenotypes

Question 33

Features of pBR322

Answer 33

• Small mol weight
• 2 sets of antibiotic resistance genes
  o Ampicillin
  o Tetracycline
• Completely sequenced: all restriction sites known
  o >20 enzymes with unique sites
  o 7 sites in TetR site
  o 3 sites in AmpR gene

Question 34

What is alpha-complementation?

Answer 34

• If lacZ gene is truncated on 5’ end then it only codes alpha fragment (inactive on its own)
• If lacZ gene is a truncation mutant with only 3’ end -> omega fragment (Inactive on its own)
• If the 2 different mutated portions are expressed in single cell they can complement each other and function together to form a functioning B-galactosidase enzyme

Question 35

Features of the PUC Plasmid

Answer 35

• Developed to speed up process of identifying recombinants
• Derived from pBR322
• All restriction sites are clustered into a multiple cloning site
• Multiple cloning site inserted into the 5’ end of truncated lacZ gene
• Is smaller than pBR322

Question 36

What does B-galactosidase do?

Answer 36

Breaks down lactose into glucose and galactose

Question 37

Identification of recombinant PUC plasmids

Answer 37

• Transformants plated onto agar & ampicillin while simultaneously screening for B-galactosidase
• X-gal in medium:
  o Lactose analogue
  o non-inducing chromogenic substrate for B-galactosidase
• IPTG (non-metabolisable lac inducer)
  ____________________
  a. Only cells with Recombinant DNA will survive due to ampicillin resistance

b. Colonies with only plasmid DNA will synthesise B-galactosidase and cleave X-gal resulting in blue product
c. Colonies with Recombinant DNA cannot synthesise B-galactosidase so X-gal isn’t cleaved so no blue product is formed

Question 38

2 methods of gene identification

Answer 38

1. Direct Screening

2. Gene bank screening

Question 39

Marker rescue

Answer 39

• Extends the scope of direct selection
• Requires availability of a mutant strain with respect to the gene in question
• Mutant strain used as host for transformation
• Requires a medium in which only the wild type strain can survive
• Introduces a marker (gene) that rescues the mutation in the host cell (i.e. codes for something the host cell is lacking)
  a. i.e. only recombinant molecules will have marker and will survive in medium.

Question 40

Hybridisation probing

Answer 40

• Any 2 single-stranded DNA mols have the potential to hybridize with each other
• Most often, the hybrid DNA molecules is unstable
  o Only a small number of individual inter-strand bonds will be formed
• If polynucleotides are complimentary (i.e. probe used has high degree of complementarity to the target gene), extensive base pairing occurs
  o Forms a stable double-stranded DNA molecule
• Nucleic acid hybridization can be used to identify a specific rDNA clone
  o Requires availability of DNA/RNA probe complimentary to the desired gene
• NaOH conc is 0.4N (equivalent/L); renders ALL DNA single-stranded due to covalent bonds between primary amino groups in nylon membrane and thymidine residue in DNA
• Proteases degrade protein, all that is left is naked DNA
• Probe added, then hybridisation found using autoradiography

Question 41

Requirements of a good probe in hybridisation probing

Answer 41

1. Must share homology with part of the gene of interest

2. Must have NO homology to vector sequences

Question 42

How can you generate a DNA probe for hybridisation probing?

Answer 42

1. Amino acid sequence (or partial sequence) of protein coded for by gene of interest is known
   a. Often the protein has been studied in some detail
   b. Can use the genetic code to predict the nucleotide sequence of the relevant gene
   c. Problem is that prediction will always be approximate due to the degeneracy of the genetic code
   d. You look for amino acid sequences (of 6 residues) that contain tryptophan and methionine [they the only 2 that have a single codon that codes for them]
   e. The other 4 residues should have as few codon combinations as possible -> ±18 different possible nucleotide sequences
   f. The oligonucleotide specific sequences can be synthesized in the lab
   g. Reprobing can be done by oligonucleotide sequences from different portions of the protein amino acid sequence
2. The gene is a member of a family of related genes
   a. Often significant nucleotide homology occurs between genes from different organisms that code for the same protein
   b. Conserved structure/sequence reflects conservation of gene function during evolution
   c. Although the DNA mols will not be entirely complementary, enough base pairing will be formed to generate a stable hybrid.

Question 43

Immunoscreening

Answer 43

• Immunological method used to detect proteins coded by the gene of interest
• This method presupposes that:
  o Cloned gene is expressed
  o Gene product not normally present in host cell
• Antibodies are required
  o If a purified protein sample is available it can be inoculated into a rabbit which can raise antibodies for it
  o Antibody will only bind to protein against which it was raised
• Protein A, G, L are native recombinant proteins of microbial origins that bind to mamalian immunoglobulin molecules

Question 44

Problem with immunoscreening and it solution

Answer 44

• Problem wiith immunological screening is that a gene is not often expressed in a foreign organims
  o It is unlikely that cloned animal/plant genes are expressed in E coli
• You may circumvent the problem by cloning in the expression vectors where the foreign gene is placed under the contol of E. coli expression signals present on the vector