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The Polymerase Chain Reaction: Development of a Practical Illustrating its Use

ZOE SMITH and C DAVID O'CONNOR l

Department o f Biochemistry University o f Southampton Basset Crescent East Southampton S 0 9 3TU, UK

Introduction The polymerase chain reaction [PCR] is a powerful technique that has revolutionized the way in which DNA analysis is carried out. 1-3 In addition to establishing itself as a fundamental molecular genetic and biochemical tool, the procedure is now increasingly used in clinical diagnosis and is likely to increase in importance for the forseeable future. Given these consider- ations, and the fact that increased demand has led to reductions in the cost of PCR reagents, it is now appropriate to have a practical that teaches undergraduate students the basic principles and applications of the method.

Our aim was to devise a practical that would illustrate the important features of PCR and produce unambiguous data for further analysis by students. Moreover, it was also important that the practical should be cost-effective [<£3 per student] and have optimized reaction conditions to facilitate success in students' hands. The final version of the practical fulfills these criteria and is now in regular use at second-year undergraduate level at Southampton•

Materials and Methods The Escherichia coli K-12 strains used were: W3110 (inv rrnD- rrnE), 4 JM83 (ara, Alac-pro, strA, thi, dp8OdlacZ AM15) 5 and CA8224.1 (Hfr lacZp4037(L37), lacZp4105 (= UVS), relA1, spoT1, thi-l). 6'7

Preparation of DNA Oligonucleotides were made on an ABI synthesizer on a 0.2 ~mole scale and purified using OPC cartridges as recommended by the manufacturer. Genomic DNA was prepared as described 8 and further purified by CsCI ultracentrifugation.9

PCR 5 × concentrated primer/reaction buffers were prepared containing the appropriate combination of primers (4 mM of each) and 50 mM Tris-HC1 (pH 8.8 at 25°C) containing 250 mM KC1, 10 mM MgC12, 0.5% (v/v) Triton X-100. The final PCR mixture consisted of wild-type or mutant genomic DNA (10- 25 ng), dNTPs (50 p~mol of each, added from a 2 mM stock solution) and the appropriate 5 × primer/reaction buffer (20 p.l). Sterile distilled water was then used to bring the mixture to 100 Ixl and mineral oil (50 Ixl) added. The tubes were held at 95°C for 5 rain prior to the addition of Taq polymerase (2.5 units), spun briefly and then incubated on a Techne PHC-1 thermal cycler as follows: 94°C, 2 min; 63°C, 2 rain; 74°C 2 min. After 30 such cycles, CHC13 (100 Ixl) was added to each tube and, after mixing and a brief centrifuge spin, 50 ixl of the aqueous phase was transferred to a fresh tube. Gel loading buffer (10 i~l) was added and the samples analyzed by electro- phoresis through 0.8% (w/v) agarose gels. 9

Results Design of the Practical A key feature of PCR is its ability to discriminate between specific wild-type and mutant DNA sequences in a genome. To illustrate this principle we used the lac operon of E coil, for which a wide range of well-characterized mutations are known. By designing suitable oligonucleotide primers it should be possible to distinguish between the wild- type and mutant lac sequences in genomic DNA (Fig 1).

Lac 2 lacUV5 mutation lacZ delta Mi5 mutation

Lac 1 OR Lac UV5

I 1,984 BASE PAIRS

1,852 BASE PAIRS

P R I M E R S E Q U E N C E S

Lacl:5'-AAACCGCCTCTCCCCGC-3'

Lac2:5'-CGCCAAAATCACCGCCG-3'

LacUV5:5'-CACI-I-i'ATGCTTCCGGCTCGTATAA-3'

I . tact gene P = ~omoter ~ lac operon O = lac operator Z = lacZ gene Y = lacY gone

Figure 1 Schematic representation of the lac operon showing the relative positions of the lac mutations and the binding sites for the PCR primers used in this work. The locations of the lacUV5 and AM15 mutations are indicated as are the Lacl, Lac2 and LacUV5 primer binding sites (not to scale). PCR amplification using Lacl + Lac2 produces a 1984 base pair fragment whereas amplification using LacUV5 + Lac2 produces a 1852 base pair fragment

Additionally, the relative positions of the primer binding sites in the lac operon region can be estimated by determining the sizes of the DNA fragments amplified following PCR. These may subsequently be compared with the fragment sizes predicted from the DNA sequence of the operon.

Design and use of Lacl and Lac2 Primers Two 17 base primers, permitting the amplification of both wild-type and mutant lac operon sequences, were first designed (Fig 1). Lacl is comple- mentary to a region on the upper strand in the lacl gene whereas Lae2 is complementary to a region on the lower strand in the lacZ gene (positions 1034-1050 and 3002-3018 of the assembled lac sequences, respectively). Both primers lack internal com- plementarity and do not form 'primer-dimers'.

PCR of E coli genomic DNA from a strain with the wild-type lac operon was carried out using the Lacl and Lac2 primers. As expected, a 1984 base pair fragment was specifically amplified (Fig 2). Further experiments with different annealing tempera- tures and magnesium ion concentrations established that the

o 2 + optimal conditions were 61 C and 2 mM Mg , respectively. DNAs from strains carrying either the lacZAM15 deletion 5

or the lacUV5 double-point mutation, 6"7'1° were then used as templates for amplification using the same primers. While specific DNA fragments were obtained in both cases, the AM15 deletion caused only a slight decrease in the size of the amplified fragment (Fig 2), consistent with the report N that the cor- responding protein lacks amino acid residues 11-41. As it would be difficult for a student to determine the extent of the deletion by gel electrophoresis of the amplified fragments, this mutant strain was not included in subsequent PCR studies.

Design and use of primers for the detection of the LacUV5 mutation The lacUV5 mutant carries a double-point mutation in the - 1 0 region of the promoter for the lac operon [5' • . . T A T G T F . . . - - ~ . . . T A T A A T . . . ] , leading to increased transcription of the lac operon in the absence of activation by the Catabolite Activator Protein. We therefore designed a 19-base primer that was a perfect match to the lacUV sequence to see if it could be used to distinguish between templates containing the wild-type and mutant sequences. We found that the predicted 1852 base pair fragment could be amplified by PCR in conjunction with the Lac2 primer. However, the optimal annealing temperature for the reaction was about 10°C lower

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1.984 kb

Figure 2 Detection of the lacZAM15 deletion using the Lacl + Lac2 primer combination. Lane 1, IacZAM15 DNA template with Lacl + Lac2 primers; lane 2, wild-type DNA template with Lacl + Lac2 primers. The DNA size markers used (M) were from BRL ("1 kb ladder"). The position of the 1984 base pair fragment of wild-type genomic DNA amplified by the Lacl + Lac2 primers is indicated

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than for the Lacl + Lac2 reaction. This meant that it would be difficult for students to carry out both PCR amplifications simultaneously on the same thermal cycler.

To circumvent this problem, a computer program, "OLIGO", 12 was used to design a longer PCR primer specific for the lacUV5 sequence to allow a higher annealing temperature to be employed. The new primer was 6 bases longer than the original primer and was termed LacUV5. Fig 3 shows that, using an annealing temperature of 63°C, both the Lacl + Lac2 and LacUV5 + Lac2 primer combinations could be used simul- taneously. Under the optimized conditions, the lacUV5 primer successfully discriminated between the wild-type and mutant lac DNA sequences whereas the Lacl + Lac2 primers amplified specific fragments from both wild-type and mutant genomic DNA templates. Good yields of product were also obtained with <5 ng of template DNA (Fig 4), indicating that variations in the [template DNA] would not affect the successful amplication of specific fragments.

The final protocol, in which the number of operational steps has been minimized, therefore used both the Lacl + Lac2 and LacUV5 + Lac2 primer combinations to allow students to distinguish between genomic DNAs carrying wild-type or mutant lac operons. The students were also asked to determine the sizes of the amplified fragments, following the construction of a standard curve from molecular size standards, and to predict what would happen if the annealing temperature was too high or too low [Copies of the actual protocol are available from the authors on request].

¸̧1,̧¸̧ ̧ i

¸̧ :

Figure 3 PCR amplification of specific wild-type and mutant lac sequences using the Lacl + Lac2 or LacUV5 + Lac2 primer combinations. Lane 1, wild-type E coli DNA template with Lacl + Lac2 primers; lane 2, wild-type E coli DNA template with LacUV5 + Lac2 primers; lane 3, lacUV5 E coli DNA template with Lacl + Lac2 primers; lane 4, lacUV5 E coli DNA template with LacUV5 + Lac2 primers; lane C, no template DNA with Lacl + Lac2 primers. The sizes of the DNA markers [BRL "1 kb ladder"] in the relevant region of the gel are indicated in kilobases

Figure 4 Testing the effect of[template DNA] on the amplification of a specific lac fragment. Lane 1, 10 ng of lacUV5 DNA template with Lacl + Lac2 primers; lane 2, 5 ng oflacUV5 DNA template with Lacl + Lac2 primers; lane 3, 1 ng oflacUV5 DNA template with Lacl + Lac2 primers; lane M, DNA size markers (BRL "'l kb ladder")

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Concluding Remarks The PCR practical that has been developed has been introduced into our Second-Year Biochemistry practical class. 69 out of the 99 students in the class obtained the predicted amplified D N A fragments following PCR, indicating that the practical works reliably for the majority of students. The most common cause of failure (about 20% of the students) was non-amplification of the target DNA, probably due to the inadvertent omission of one of the essential PCR components. Contamination of samples with extraneous D N A has (so far) not been a problem. Comments from the students have generally been favourable and, following the practical, most seem to have an improved understanding of the principles behind PCR as well as its applications.

A c k n o w l e d g e m e n t s This work was supported by a grant from the Society for General Microbiology (Fund for Developments in Teaching). We are also indebted to Techne Ltd for their generous gift of a thermal cycler.

References Saiki, R K, Scharf, S, Faloona, F, Mullis, K B, Horn, G T, Erlich, H A and Arnheim, N (1985) Science 230, 1350-1354

2Innis, M A, Gelfand, D H, Sninsky, J J and White, T J (Editors), (1990) PCR Protocols: A guide to methods and applications, Academic Press, San Diego

3Bloch, W (1991) Biochemistry 30, 2735-2747 4Kohara, Y, Akiyama, K and Isono, K (1987) Cell 50, 495-508 5Vieira, J and Messing, J (1982) Gene 19, 259-268 6Arditti, R R, Scaife, J G and Beckwith, J R (1968) J Mol Biol 38,

421-426 7Silverstone, A E, Arditti, R R and Magasanik, B (1970) Proc Natl Acad Sci USA 66, 773-779

sSilhavy, T J, Berman, M L and Enquist, L W (1984) Experiments with Gene Fusions, Cold Spring Harbor Press, Cold Spring Harbor, New York

9Maniatis, T, Fritsch, E F and Sambrook, J (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York

l°Beckwith, J R (1965) J Mol Biol 8, 427-430 11Zabin, I and Fowler, A V (1978) in The Operon (edited by Miller, J H

and Reznikoff, W S), pp 89-121, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

12Rychlik, W and Rhoads, R E (1989) Nucleic Acids Res 17, 8543-8551

A ldose Ketose No chiral No No chiral No

centers stereoisomers centers stereoisomers

Tetrose 2 4 l 2 Pentose 3 8 2 4 Hexose 4 16 3 8 Heptose 5 32 4 16

Total stereoisomers 60 30

In other words, there are 90 possible D and L forms of the specified carbohydrates; hence, 45 and not 40, D forms.

The author attributes 'configuration' to achiral carbon atoms on several occasions (C2 of xylulose and sedoheptulose and for the completely achiral dihydroxyacetone). While this term has proved hard to define in general, in carbohydrate chemistry 'configurational differences are usually associated with different spatial arrangements for tetrahedrally disposed ligands attached to chiral carbon atoms'. 2 To take only one example, it would have been preferable to refer to the configurations at C2 and C3 of ribose and at C3 for xylulose and sedoheptulose rather than 'the configuration at C2 and C3 for ribose, xylulose and sedoheptulose' .

It is stated that "fructose is the 'keto form' of glucose", although, of course, glucose does not exist as a keto structure. More precisely, fructose should be described as the ketose derived by aldose-ketose isomerization of glucose. Pyruvate is also described as ' the keto acid of L-alanine'; this statement neglects the fact that pyruvate is also the keto acid corresponding to D-alanine.

Ronald Bentley Department o f Biological Sciences

University o f Pittsburgh Pittsburgh, PA 15260, USA

References 1Chude, O (1991) Biochem Educ 19, 195-196 2Stoddard, J F (1971) 'Stereochemistry of Carbohydrates', Wiley- Interscience, New York, p 1

C h e m i c a l R a t i o n a l e f o r the Ci t r ic A c i d C y c l e

L e t t e r s to t h e E d i t o r

Using Glucose as a Focus f o r Understanding Carbohydrate

Structure and Me tabo l i sm

Dear Sir, Carbohydrate chemistry is not every student's 'cup of tea' , so Obi Chude is to be commended for pointing out structural relation- ships to facilitate memorization of the ' top ten' carbohydrates) Unhappily this presentation contains one serious error and some infelicitous phrases.

Reference is made to ' the forty O sugars (containing 4 to 7 carbon atoms) derived from D-glyceraldehyde and dihydroxy- acetone'. For the unsymmetrical structures under consideration, the presence of n chiral centers leads to 2" stereoisomers. Hence, the total number of D and L forms can be derived as follows:

Dear Sir, When discussing the chemical rationale for the citric acid cycle P D J Weitzman claims that the strategy for oxidising acetic a c i d . . , depends on its combination with another molecule of acetic acid to form succinic acid". He continues "the first section of the cycle is designed to produce succinic acid; the remaining section brings about oxidation of what was originally a methyl group of acetic acid". In my opinion neither of these statements is correct and both will serve to confuse students about the structure of the cycle.

To a large extent it is fruitless to speculate about the rationale for a metabolic pathway; one cannot question evolution. The only valid rationale for a pathway is that it works! On the other hand one can, and of course should, enquire as to how a pathway works; the citric acid cycle works as follows.

There is no chemical precedent to indicate that the carbons in acetate can be converted directly to carbon dioxide other than by combustion. This problem is overcome in the citric acid cycle by incorporating both carbons of acetate into a structure (enzyme bound oxalosuccinate) which allows two molecules of carbon dioxide to be liberated by two successive oxidative decarboxyl- ations. At this stage in the cycle (succinyl CoA) the oxidation of the carbons of acetate is complete. It is irrelevant that, as shown by labelling studies, the carbons liberated as carbon dioxide are

B I O C H E M I C A L E D U C A T I O N 20(3) 1992