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Several different polymerase chain reactions (PGR) have been used to amplify polymorphisms in the Interleukin-6 (EL6), p-Fibrinogen and C-reactive protein (CRP) genes, however the same principles apply regardless of the specifics of the reaction used. The primer sequences and PCR cycle conditions specific to each PGR are detailed below in Tables 2 and 3. The components of each PCR and restriction enzyme digest mix are listed in Table 4.

In brief, the technique of PCR relies on the fact that the DNA strand is denatured into single strands by heat, and will anneal with primers and nucleotide bases to reform a double strand on cooling. The first step is therefore a short high temperature period to denature the DNA. This initial stage is followed by cooling in the presence of primers that are complementary to the DNA either side of the sequence to be studied. These primers anneal, and a DNA polymerase adds nucleotides base by base, thus replicating the DNA. The polymerase used is derived from the bacterium Thermus aquaticus (Taq)

and is heat stable. It therefore does not need to be replenished after each cycle of heating and cooling. This technique allows very small initial amounts of DNA to be increased in quantity until there is sufficient DNA for genotyping to be performed.

Restriction enzymes are enzymes derived from bacteria that will cleave double stranded DNA at a particular sequence. The enzyme translocates along the DNA until a particular recognition site is reached. At this point the DNA is cut. The restriction enzyme is sensitive even to a single base change in the recognition sequence, and thus can be used

to detect point mutations and single base polymorphisms. A single base change can either eliminate or create a cutting site for a particular enzyme.

2.2.7 Sample Preparation For Polymerase Chain Reaction

Following extraction and dispensing into working 96-well arrays, DNA samples were prepared for PCR by centrifuging the DNA working-array at 1500g for 1 minute. This was to ensure that all the DNA dilutions were at the bottom of their respective wells, thus reducing the possibility of cross-well contamination when the array lid was removed.

Two and a half microlitres of each sample was then removed from each array and transferred into a standard 96-well PCR plate (Omniplate, Hybaid) using a Finnipipette multichannel dispenser (Life Sciences, Basingstoke, Hants, UK). Positive and negative controls were utilised to ensure accuracy. Extreme care was taken to ensure that samples were place in the identical orientation as in the original arrays. Loaded Omniplates were then centrifuged at 1500g for 30 seconds to ensure that the DNA was at the bottom of each well, and then dried on a Thermal Cycler block (MJ Tetrad DNA Engine Thermocycler) at 90° for 10 minutes.

A bulk-mix of reagents was made up for each PCR, allowing adequate volume for the planned number reactions with an additional 10% added to ensure that the mix would not run short. PCR primers and Taq polymerase (Gibco-BLR LTd., Paisley UK) were kept on ice and added last.

Polymerase chain reaction were performed in a total volume of 20|xl. Each reaction contained Ix concentration of polmix (50mM KCl, lOmM Tris-HCl (pH 8.3), 0.2mM dATP, dGTP, dTTP and dCTP) or 1 x NH3 buffer (16mM (NH4)2S04, 67mM Tris-HCl (pH 8.4), 0.01% Tween 20, 0.2mM of each dNTP) and MgCl2, 10 pmol of each primer and 0.3U of Taq polymerase. Magnesium concentration varied with each PCR.

The PCR mix was added to each well of the PCR plate using an automatic Biohit repeating dispenser (Alpha Laboratories, UK). Each sample was overlaid with of

mineral oil to prevent evaporation. The microtitre plate was then sealed with clear sticky plastic lid and carefully labelled. Plates were centrifuged at lOOOrpm for 30 seconds to ensure good mixing of the reaction components in each well. PCR amplification was performed on an MJ Tetrad DNA Engine Thermocycler, using cycle conditions specific to each PCR.

A bulk mix of restriction enzyme digest mix was made up in a large Bppendorf tube on each occasion, containing sufficient enzyme to digest the PCR products in each well of the PCR plate. 5pi of digestion mix was then added to lOpl of each reaction product using a repeater pipette as for the PCR mix. Each omniplate was then centrifuged at 1000 rpm for 30 seconds to ensure that the PCR product and restriction enzyme mixed well. The PCR/digestion mix was then incubated overnight at 37°. The specific details for each PCR and digestion mix are detailed in Table 4.

2.2.2.1 IL6 -174 G>C Promoter Polymorphism

Fishman et al originally identified the presence of a single base mutation at position - 174 in the IL6 gene promoter where a cytosine (C) residue is substituted for a guanine (G) - recorded as -174G>C (Fishman, Faulds et al. 1998). PCR generates a 190 base pair fragment in the DNA sequence flanking the site of the polymorphism.

DNA fragments generated by PCR were digested using the restriction enzyme N lalll

(New England Biolabs). The rare cutting C-allele will produce two fragments of sizes 143 and 47, whereas the non-cutting wild-type G-allele will remain as an uncleaved 190 base pair fragment.

2.2.2.2 IL6 Promoter -572G>C And -597G>A Polymorphisms

Two additional IL6 gene promoter polymorphisms have been identified within the EL6 gene promoter: -572G>C (a guanine for cytosine base substitution) and -597G>A (guanine for adenine) (Terry, Loukaci et al. 2000; Humphries, Luong et al. 2001). The - 597G>A and -572 G>C polymorphisms genotypes are in close proximity and were determined using the same PCR primers, generating a 163 bp product.

Following PCR 10)li1 of PCR product was removed from each well (i.e. separating the PCR product into two) and placed in a new round-bottom digest plate. This was to enable restriction enzyme digestion to be completed for both the -572G>C and - 597G>A polymorphisms from the same PCR product. The rare -597A allele creates a

Fok I restriction cutting-site, yielding fragments of 116 and 47bp, whilst the -597G- allele produces an uncut PCR fragment of 163bp. The -572G-allele creates a digestion site for M bil, yielding fragments of 101 and 62 bp. The -572A-allele is uncut, hence a fragment of 163 bp.

2.2.3.1 P-Fibrinogen -455 G>A Promoter Polymorphism

The p-fibrinogen promoter has a common polymorphism at position -455 where there is a guanine for adenine base substitution, denoted -455 G>A (Thomas, Lamlum et al. 1994). PCR generates a fragment of 176 bp, with the wild-type G-allele creating a cutting site for Hae III (or its isoschizomer, BSuRl) producing two fragments of 98 and 76bp. The -455A-allele remains uncut, producing a fragment of 176bp.

2.2.3.2 j3-Fibrinogen -854 G>A Promoter Polymorphism

A second promoter polymorphism has been identified at position -854, with a G to A base substitution (Behague, Poirier et al. 1996). Here the presence of the rare -854A- allele generates a cutting site for the restriction enzyme Taql, producing fragments of 79 and 23bp, whilst the common G-allele is uncut (102bp fragment).

2.2.4.1 CRP Promoter -748A>Polymorphism

Using SSCP a novel single nucleotide polymorphism has recently been identified at position -748 in the CRP gene promoter by Dr Franco Zito (unpublished data 2000). At this site there is an adenine for guanine base substitution. PCR generates a fragment of 136bp that is cleaved by the restriction enzyme SacII in the presence of the rare -748A- allele, producing fragments of 113 and 23bp.

2 2 .4 .2 CRP Exon-2 -\-1059G>C Polym orphism

The first CRP polymorphism was identified in exon-2 of the CRP gene, at position +1059 where there is a guanine for cytosine base substitution (Cao and Hegele 2000). Using the published DNA sequences, PCR was performed generating a DNA fragment of 94bp. The restriction enzyme FNU4 cleaves the DNA in the presence of the common G-allele, generating fragments of 73 and 21bp, whilst the rare C-allele is uncut (94bp).

2.2.4.3 CRP 3 ' UTR + 1 4 4 4 0 T Polymorphism

An additional polymorphism has been identified in the 3’untranslated region (UTR) of the CRP gene at position +1444 (Dr Aroon Hingorani, personal communication). At this position there is a thymidine for cytosine base substitution. PCR was used to generate a 181bp fragment of DNA. The common C-allele is cleaved by the enzyme Bsp 12861

yielding fragments of 158 and 23bp, whilst the rare T-allele remains uncleaved (181bp).