Recombinant DNA technology: Lecture 1

Background reading:

Please see the ReBase website for information regarding Restriction Enzymes: http://rebase.neb.com/rebase/rebhelp.html

For Restriction Mapping, click on this link: Restriction Mapping Page
For Additional Help, click on this link: Helpful Hints

How do we obtain DNA and how do we manipulate DNA?

1. Remove tissue from organism
2. Homogenise in lysis buffer containing guanidine thiocyanate (denatures proteins)
3. Mix with phenol/chloroform - removes proteins
4. Keep aqueous phase (contains DNA)
5. Add alcohol (ethanol or isopropanol) to precipitate DNA from solution
6. Collect DNA pellet by centrifugation
7. Dry DNA pellet and resuspend in buffer
8. Store at 4°C

It is often necessary to 'break up' large DNA molecules into smaller, more manageable fragments - often to sizes ranging from 100 bp to 2 kb (bear in mind that each resulting DNA fragment is an individual molecule). These smaller fragments can then be manipulated more easily - to isolate particular DNA fragments, to characterise their molecular sequence, to determine their function, to determine their position in relation to other sequences within the genome, to use them to express proteins, etc. .

How do we manipulate DNA?

It used to be difficult to isolate enough of a particular DNA sequence to carry out further manipulation and/or characterisation of its molecular sequence. DNA is a macromolecule - it is made up of a sequence of lots and lots of deoxyribonucleotides. Large DNA molecules can be fragmented using 'shearing' forces, in other words mechanical stress to 'shred it', thus creating smaller fragments. However, the resulting fragmentation is not reproducible - the breakage points can occur anywhere within the molecule, thus each DNA molecule will be randomly broken down and various different-sized fragments can be generated, any of which can have the DNA sequence of interest. A further difficulty in isolating a particular DNA fragment is that standard chemical/biochemical methods are not sufficient to distinguish any part of the genome from another (after all one DNA molecule is chemically similar to another).

Progress in understanding genetic mechanisms at the molecular level was slow. Then came the discovery of various bacterial and viral enzymes which modify and synthesise nucleic acids (DNA and RNA), along with the means to produce more outwith the organism from which they were originally isolated. The application of these enzymes for manipulating DNA (no matter what the source) led to the creation of Recombinant DNA Technology which has enabled great scientific advances in the field of biology, has created new scientific disciplines and has revolutionised our world.

Until 1970 there were no convenient methods available for cutting DNA into discrete, manageable fragments.

1970 - The Beginning of the Revolution
Discovery of a restriction enzyme in the bacterium Haemophilus influenzae

Restriction enzymes are endonucleases

• Bacterial enzymes
• Different bacterial strains express different restriction enzymes
• The names of restriction enzymes are derived from the name of the bacterial strain they are isolated from
• Cut (hydrolyse) DNA into defined and REPRODUCIBLE fragments
• Basic tools of gene cloning

Names of restriction endonucleases

EcoRI -  from Escherichia coli
BamHI - from Bacillus amyloliquefaciens
HindIII - from Haemophilus influenzae
PstI -  from Providencia stuartii
Sau3AI - from Staphylococcus aureus
AvaI -  from Anabaena variabilis

Restriction enzymes recognise a specific short nucleotide sequence

This is known as a Restriction Site

The phosphodiester bond is cleaved between specific bases, one on each DNA strand

The product of each reaction is two double stranded DNA fragments

Restriction enzymes do not discriminate between DNA from different organisms

Restriction endonucleases are a natural part of the bacterial defence system

• Part of the restriction/modification system found in many bacteria
• These enzymes RESTRICT the ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host bacterial cell by cutting it up (degrading it)
• The host DNA is MODIFIED by METHYLATION of the sequences these enzymes recognise
• Methyl groups are added to C or A nucleotides in order to protect the bacterial host DNA from degradation by its own enzymes

Fig 7-5b, Lodish et al (4th ed)

Types of restriction enzymes

• Type I  Recognise specific sequences·but then track along DNA (~1000-5000 bases) before cutting one of the strands and releasing a number of nucleotides (~75) where the cut is made. A second molecule of the endonuclease is required to cut the 2nd strand of the DNA
• e.g. EcoK.
• Require Mg²⁺, ATP and SAM (S-adenosyl methionine) cofactors for function
• Type II  Recognise a specific target sequence in DNA, and then break the DNA (both strands), within or close to, the recognition site
• e.g. EcoRI
• Usually require Mg²⁺
• Type III  Intermediate properties between type I and type II. Break both DNA strands at a defined distance from a recognition site
• e.g. HgaI
• Require Mg²⁺ and ATP

Hundreds of restriction enzymes have been isolated and characterised

• Enables DNA to be cut into discrete, manageable fragments
• Type II enzymes are those used in the vast majority of molecular biology techniques
• Many are now commercially available

Each restriction enzyme will recognise its own particular site

• Some recognise very short sequences consisting of only 4 base pairs. These tend to cut DNA more frequently (generating smaller fragments) as the likelihood that any stretch of DNA sequence will contain these minimal recognition sites is high.
 approximately 1 site per 256 bases ([1/4]⁴)
• Some require longer recognition sequences (up to 8 bp). The longer the recognition sequence the less frequently these sites are likely to occur in any particular DNA sequence. Enzymes which cut DNA very infrequently are known as RARE cutters.
 an 8 bp recognition site will occur approximately 1 per 65,536 bases ([1/4]⁸)

Some recognise more than one sequence

• There are restriction enzymes which allow substitutions in one or more positions of their recognition sequences.
• Most common substitutions
• purines (A or G), designated R
• pyrimidines (C or T), designated Y
• any nucleotide, designated N

The consensus HincII recognition site is designated 5'-G T Y R A C-3'
 

Many Type II restriction endonucleases recognise PALINDROMIC sequences

• Symmetrical sequences which read in the same order of nucleotide bases on each strand of DNA (always read 5'g3')

The high specificity for their recognition site means that DNA will be cut reproducibly into defined fragments

• Generate restriction maps
• Isolate and clone specific DNA fragments

Different enzymes cut at different positions and can create single stranded ends ('sticky ends')

• Some generate 5' overhangs - eg: EcoRI

• Some generate 3' overhangs - eg: PstI

• Some generate blunt ends - eg: SmaI

The 'sticky' overhangs are known as COHESIVE ENDS

• The single stranded termini (or ends) can base pair (ANNEAL) with any complementary single stranded termini

• Inserting foreign DNA into a cloning vector

Restriction enzymes are a useful tool for analysing Recombinant DNA

• Checking the size of the insert
• Checking the orientation of the insert
• Determining pattern of restriction sites within insert DNA

 Electrophoresis
Linear DNA fragments of different sizes are resolved according to their size through gels made of polymeric materials such as polyacrylamide and agarose. For instance, agarose is a polysaccharide derived from seaweed - and gels formed from between 0.5% to 2% (mass/volume i.e. 0.5 to 2.0g agarose/100 ml of aqueous buffer) can be used to separate (resolve) most sizes of DNA

DNA is electrophoresed through the agarose gel from the cathode (negative) to the anode (positive) when a voltage is applied, due to the net negative charge carried on DNA

When the DNA has been electrophoresed, the gel is stained in a solution containing the chemical ethidium bromide. This compound binds tightly to DNA (DNA chelator) and fluoresces strongly under UV light - allowing the visualisation and detection of the DNA.

Like any molecule that binds to DNA, ethidium bromide is hazardous. It is a mutagen. Always wear gloves when working with ethidium bromide.
 
 

Remember - not all enzymes used for Recombinant DNA Technology are restriction enzymes

Other useful DNA modification enzymes used for manipulating DNA:

Alkaline phosphatase	Removes phosphate groups from 5' ends of DNA (prevents unwanted re-ligation of cut DNA)
DNA ligase	Joins compatible ends of DNA fragments (blunt/blunt or complementary cohesive ends). Uses ATP
DNA polymerase I	Synthesises DNA complementary to a DNA template in the 5'-to-3'direction. Starts from an oligonucleotide primer with a 3' OH end
Exonuclease III	Digests nucleotides progressiviely from a DNA strand in the 3' -to-5' direction
Polynucleotide kinase	Adds a phosphate group to the 5' end of double- or single-stranded DNA or RNA. Uses ATP
RNase A	Nuclease which digests RNA, not DNA
Taq DNA polymerase	Heat-stable DNA polymerase isolated from a thermostable microbe (Thermus aquaticus)

webpage last updated 20/2/03

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