Molecular Biology:

1. Polymerase Chain Reaction (PCR) and Its Variations

2. Electrophoresis (RLFP analysis)

- Agarose

- SDS –PAGE

- Pulsed Field Gel Electrophoresis

3. DNA Cloning

- Restriction endonucleases

- Restriction map

- Cloning Vectors

- Identification of Cloned DNA

4. Construction of DNA Library

5. Hybridization

6. DNA Microarray

7. Phage Display

8. Typing Methods (RFLP, AFLP)

BB211:  Cell and Molecular Biology Dr Eve Lutz Department of Bioscience

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Recombinant DNA technology: Lecture 1

Introduction to Recombinant DNA technology Restriction enzymes and gel electrophoresis

Background reading:

Chapter 10 pp 205-206; Chapter 16 Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

An organism's genome contains virtually ALL the information necessary for its growth and development

Determining the molecular sequence of DNA that makes up the genome of different organisms is an international scientific goal, several laboratories are participating worldwide in this task (including the Wellcome Trust Sanger Institute  and the Roslin Institute here in Britain). It is thought that having access to the complete DNA sequence of an organism can help us not only to decipher its biology but also help us understand major biological questions, for instance, what makes one species pathogenic whereas a related species is not. Or what are the genetic mechanisms which lead to disease and can they be reversed, halted or even prevented? It has the potential to help us understand very complex biological processes which are dependent on the interaction of a number of different genes (like development or the transmission and progression of particular diseases) and which have multifactoral causes and effects. And of course, in the day to day world, we use particular gene products (proteins and enzymes, peptides) that have been 'adapted' for commercial use (therapeutics, laundry detergents, genetically modified organisms such as tomatoes, rice, sheep etc etc).

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

Quite straightforward to isolate DNA For instance, to isolate genomic 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

Each cell (with a few exceptions) carries a copy of the DNA sequences which make up the organism's genome. However, many genomes are large and complex (for instance the human genome is made up of ~3000 x 106 base pairs). A particular DNA sequence (for instance the allele of a gene) can be very small in comparison. And it probably occurs only once or twice within the genome (ie only one or two copies per cell). This means that a particular DNA sequence will be present as only a (very) small part within the complex mixture of DNA sequences that make up the genomic DNA of that organism.

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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.

Recombinant DNA Technology

Techniques for                     - Isolation                     - Digestion                     - Fractionation                     - Purification of the TARGET fragment                     - Cloning into vectors                     - Transformation of host cell and selection                     - Replication                     - Analysis                     - Expression of DNA

DNA is manipulated using various enzymes  that modify and/or synthesise it

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

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Restriction enzymes

Enzymes that can cut (hydrolyse) DNA duplex at specific sites. Current DNA technology is totally dependent on restriction enzymes.

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

Titles of restriction enzymes are derived from the first letter of the genus + the first two letters of the species of organism from which they were isolated.

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

Most restriction enzymes will cut DNA which contains their recognition sequence, no matter the source of the DNA

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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

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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)

The sites occur more randomly than predicted, so that digestion by any one enzyme will generate DNA fragments of different lengths

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

For example HincII will allow two substitutions in each of two sites. It recognises and cuts 4 different sequences.

5'-G T C GA C-3'      5'-G T T G A C-3'      5'-G T C A A C-3'      5'-G T T A A C-3' 3'-C A G C T G-5'      3'-C A A C T G-5'       3'-C A G T T G-5'     3'-C A A T T G-5'

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 5'3')

For example, EcoRI recognises the sequence 5'-G A A T T C-3'

3'-C T T A A G-5'

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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

Examples of restriction enzymes and the sequences they cleave

Source microorganism EnzymeRecognition SiteEnds produced

Arthrobacter luteus AGCT

Bacillus amyloiquefaciens H HI GGATCC

Escherichia coli RI GAATTC

Haemophilus gallinarum GACGC(N)

Haemophilus infulenzae III AAGCTT

Providencia stuartii 164 CTGCANocardia otitiscaviaruns GGCCGC

Staphylococcus aureus 3A 3A GATC

Serratia marcesans CCCGGG

Thermus aquaticus TCGA

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The 'sticky' overhangs are known as COHESIVE ENDS

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

This is the basis for RECOMBINANT DNA TECHNOLOGY Inserting foreign DNA into a cloning vector

Restriction enzymes are a useful tool for analysing Recombinant DNA

After ligating a particular DNA sequence into a cloning vector, it is necessary to check that the correct fragment has been taken up. Sometimes it is also necessary to ensure that the foreign DNA sequence is in a certain orientation relative to sequences present in the cloning vector.

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

DNA fractionationSeparation of DNA fragments in order to isolate and analyse DNA cut by restriction enzymes

 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.

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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 phosphataseRemoves phosphate groups from 5' ends of DNA (prevents unwanted

re-ligation of cut DNA)

DNA ligaseJoins compatible ends of DNA fragments (blunt/blunt or complementary

cohesive ends). Uses ATP

DNA polymerase ISynthesises DNA complementary to a DNA template in the 5'-to-3'direction. Starts from an oligonucleotide primer with a 3' OH end

Exonuclease IIIDigests nucleotides progressiviely from a DNA strand in the 3' -to-5'

direction

Polynucleotide kinaseAdds 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)

Recombinant DNA: Plasmids, cloningBackground reading: Reference for this lecture please read the following: Chapter 16 Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

What is DNA cloning?

DNA cloning is the isolation of a fragment or fragments of DNA from an organism and placing in a VECTOR that replicates independently of chromosomal DNA. The RECOMBINANT DNA is propagated in a host organism, the resulting CLONES are a set of genetically identical organisms which contain the recombinant DNA

Why is DNA cloning important?

DNA cloning is involved in a number of applications (GENETIC ENGINEERING). Many techniques for DNA isolation and manipulation have been worked out and are now routine in scientific laboratories. These techniques are now important tools that every scientist must know about.

 Three main purposes for cloning DNA

1) DNA sequencing Determining the sequence of the bases in the DNA can tell us about which proteins

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or RNAs are encoded and their sequences, which sequences control their expression (GENE PROMOTERS and other control sequences), as well as any possible mutations which might alter their function. Having access to the complete DNA sequence of an organism can help us decipher the biology of that organism.

2) Protein production Isolating the gene which encodes a desired protein (haemoglobin, interferon) may allow that gene to be over-expressed so that the protein can be produced in bulk for study or use

3) Engineering animals/plants/proteins The ability to alter the properties of proteins as well as create genetically modified animals and plants (TRANSGENICS) has lead to their use for research and for therapeutic and commercial purposes. The technology may lead to the development of new therapies for the treatment of disease (GENE THERAPY).

Cloning and Expression Vectors

Isolated DNA is cloned into VECTORS for long term storage, propagation of the DNA and for production of protein from gene(s) encoded in the DNA

What are cloning vectors?

Cloning vectors are extra-chromosomal 'replicons' of DNA which can be isolated and can replicate independently of the chromosome. Vectors usually contain a selectable marker - a gene that allows selection of cells carrying the vector e.g. by conferring resistance to a toxin. DNA of interest can be cloned into the vector and replicated in host cells, usually one which has been well characterised.

Commonly used vector systems

Bacterial plasmids Bacteriophages Cosmids Yeast artificial chromosomes (YACs) Ti plasmid (plants) Eukaryotic viruses such as baculovirus (insect cells), SV40 virus and

retroviruses.

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Plasmids are the most commonly used vector system. Several types are available for cloning of foreign DNA in the host organism Escherichia coli. Many E. coli plasmids allow the expression of proteins encoded by the cloned DNA

Bacteriophage are another common vector system used for cloning DNA. These are viruses which 'infect' E. coli. The M13 bacteriophage is a single-stranded DNA virus which replicates in E. coli in a double-stranded form that can be manipulated like a plasmid. It can be used to produce single-stranded DNA copies which are useful for DNA sequencing.

Bacteriophage is another bacteriophage which is commonly used to make DNA libraries. It allows the cloning of larger fragments of DNA than can be incorporated into plasmids.

Strategy for cloning DNA into a plasmid (or other cloning) vector

SUBCLONING

cut DNA of interest with the appropriate restriction endonuclease(s) separate fragments by gel electrophoresis purify target fragment from gel

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ligate fragment with a plasmid cut with the same restriction endonuclease(s) - ligation is performed using the enzyme T4 DNA ligase, ATP and Mg2+ ions

Subcloning an EcoRI fragment into a plasmid cloning vector

Good efficiency of ligation of foreign DNA into a vector can be achieved if both the vector and the insert DNA are cut with 2 different restriction enzymes which leave single stranded ends (cohesive ends). The DNA is ligated in only one direction, and there is only a low background of non-recombinant plasmids. If only one restriction enzyme is used to cut the vector and insert, then efficiency of ligation is lower, DNA can be inserted in two directions and tandem copies of inserts may be found. To avoid high background of non-recombinants, alkaline phosphatase is used to remove 5' phosphate groups from the cut vector to prevent self-ligation.

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If vector and insert DNA are cut with an enzyme which leaves blunt DNA ends, the background of non-recombinant plasmids can be high and the best way around the problem is to use high concentrations of both DNAs and of the DNA ligase enzyme.

Transformation is the process by which plasmids (or other DNA) can be introduced into a cell. For E. coli transformation with plasmids is quite straightforward, plasmids can be introduced by electroporation or by incubation in the presence of divalent cations (usually Ca2+) and a brief heat shock (42°C) which induces the E. coli cells to take up the foreign DNA

There are different methods to select for transformed cells. For instance, transformants can be selected as antibiotic-resistant colonies on agar plates containing antibiotic.

For E. coli transformed with plasmids, colonies grown on antibiotic-containing plates should all carry plasmids. However, this does not guarantee that the plasmid contains an insert. It is possible that the vector has re-ligated and not incorporated an insert.

 A means to determine which clones contain plasmids with inserts is to use a positive control method such as insertional inactivation. This provides a clear way of recognising recombinants from those carrying re-ligated vector.

1. two antibiotic selection and replica plating 2. colour selection: blue/white selection using the lacz gene

Analysis of clones

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One of the first steps is to identify clones carrying the recombinant plasmid, with the desired DNA insert. This can be done by 'picking' clones - choosing individual bacterial colonies in order to isolate the plasmid DNA from each of them. Single bacterial colonies are grown in culture broth containing the selection antibiotic in order to maintain the plasmid. The plasmid DNA is extracted by the standard minipreparation technique and then analysed by restriction digest. After digesting the DNA, different sized fragments are separated by agarose gel electrophoresis and the sizes determined by comparison with known DNA molecular weight markers.

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 

Sometimes it is important to determine the orientation of the DNA insert in relation to the vector sequence. This can be done simply by restriction digest using enzyme(s) which cut the vector sequence near to the insert and cut within the insert sequence (asymmetrically).

Figure of agarose gel stained with ethidium bromide and visualised with uv light

Restriction mapping

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Restriction mapping is a useful way to characterise a particular DNA molecule. It enables us to locate and isolate DNA fragments for further study and manipulation. The relative location of different restriction enzyme sites to each other are determined by enzymatic digest of the DNA with different restriction enzymes, alone and in various combinations (ee restriction enzymes web notes). The digested DNA is separated by gel electrophoresis and the fragment sizes that have been generated are used to build the 'map' of sites of the fragment. The map lets us know 'where we are' in the linear DNA macromolecule.

Remember, not all DNA is linear. For circular DNA molecules (like plasmids) restriction enzymes that cut at a single site will generate a linear molecule, which will run as a single band in gel electrophoresis.  

Single and double digests of DNA

Fig 7-24, Lodish et al. (4th ed)After digestion with each enzyme, and with both enzymes, the fragments are analysed by agarose gel electrophoresis. The separated fragments are typically visualised with ethidium bromide/UV light and the sizes of the fragments determined by comparison with standard DNA molecular weight markers.

Agarose gel electrophoresis of uncut and digested DNA,

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gel stained with ethidium bromide and visualised with UV light

M: DNA molecular weight markers U: uncut DNA E: EcoRI digested DNA H/S: HindIII + SalI digested DNA Can you determine what type of DNA is being analysed on this gel?

Another method used for restriction mapping:

Analysing partial digest of an end-labelled molecule

Fig 7-25, Lodish et al. (4th ed)

The digested DNA is separated by gel electrohoresis and the DNA fragments visualised by autoradiography (the gel is exposed to film). The positions of the multiple recognition sites for enzyme II are inferred from the lengths of the labeled pieces.

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Using restriction enzyme maps for analysing Recombinant DNA

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

Detailed restriction maps are available for each cloning vector

Checking insert and orientation by restriction mapping

the orientation of the fragment can be determined by cutting with an enzyme that recognises an internal restriction site within the insert and with one that recognises a site in the polylinker of the vector. See the agarose gel example above that shows the digest of a plasmid vector and recombinant DNA (different clones of the plasmid vector with insert in the EcoRI site but in opposing orientations).

BB206 Tutorial: 

RESTRICTION MAPPING

Let us start simply:

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Example 1. The linear DNA fragment shown here has cleavage sites for BamHI and EcoRI. In the accompanying diagram of an electrophoresis gel, indicate the positions at which bands would be found after digestion with:     a. BamHI alone     b. EcoRI alone     c. BamHI and EcoRI together

The dashed lines on the right indicate the positions which bands of 1 - 12 kb would migrate.

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Now try another one. Example 2. The circular DNA molecule shown below has cleavage sites for BamHI and EcoRI. In the accompanying diagram of an electrophoresis gel, indicate the positions at which bands would be found after digestion with:     a. BamHI alone     b. EcoRI alone     c. BamHI and EcoRI together.

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The dashed lines on the right indicate the positions which bands of 1 - 12 kb would migrate.

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First, look at the gel, and determine the size fragments that are present in each lane. I have put them in the following table  

BamHI 7 kb, 3 kb

EcoRI 7 kb, 3 kb

EcoRI + BamHI 3 kb, 2 kb

Now, use the following steps as a guide to help you

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Try this one: Example 3. A 8.9 kb circular plasmid is digested with three restriction enzymes, EcoRI, BamHI and HindIII, individually and in combination, and the resulting fragment sizes are determined by means of electrophoresis. The results are as follows:

EcoRI 8.9 kb

BamHI 6 kb. 2.9 kb

HindIII 8.9 kb

EcoRI + BamHI 6 kb, 2.4 kb, 0.5 kb

EcoRI + HindIII 7.4 kb, 1.5 kb

BamHI + HindIII 5 kb, 2.9 kb, 1 kb

EcoRI + BamHI + HindIII 5 kb, 2.4 kb, 1 kb, 0.5 kb

Draw a restriction map based on these results.

A.

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If you look carefully, you will see that these maps are MIRROR IMAGES of each other. Either would be correct because they both fit the data given in the table above (Can you think of the reason WHY there would be 2 correct answers?). The important point is that you would be able to PREDICT the fragment sizes correctly from either map.

Remember - maps are a means of orientating yourself. But they can be drawn from different perspectives.

And it is important to be able to draw maps, because they help us PREDICT fragment sizes from restriction enzyme digests, which we can then VERIFY by performing the digest of the actual DNA and running it on a gel. If you are cloning and manipulating DNA, this is an invaluable tool to help you identify and distinguish between different DNA fragments, find specific DNA fragments (and may even tell you whether you have got the right or the WRONG clone!)

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If you are feeling more adventurous, try the sample problem solving test from a previous year.

This tutorial was designed by Dr Eve Lutz. Should you need more help or information, please contact her (email eve.lutz@strath.ac.uk).

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