El enfoque cognitivo conductual.

This represents hybridization o f the probes to the DNA, and subsequent cleavage by AmpliTaq DNA polym erase, show ing separation of the fluorescent probe FA M from the quencher probe when the probe is hybridized to the target tem plate, but no separation from the non-target template. (Figure taken fro m A pplied Biosystems Ltd.)

The prim er and probe sequences for the RA N TES 403 polym orphism were as follows:

• Prim er 1 (forward) 5 ’-GAG G AC C C T CCT CA A TAA AAC A CT TTA TA A A T -3 ’ • Prim er 2 (reverse) 5 ’-A CT G A G TCT TC A AAG TTC CTG C T T -3’

• Probe 1 (VIC) CAT TAC A GA TCT TA G CTC CTT T • Probe 2 (FAM ) CAT TA C AGA T C T T A T CTC CTT T

The reagents for the TaqM an PC R reaction were as follows;

Reagents Volum eAVell

(12.5ul vol reaction)

Final

Concentration TaqM an® U niversal PCR M aster

M ix 2X 6.25 ul IX

40X Assay Mix 0.3 ul IX

G enom ic DNA 2ul

H2O 3.9 ul

Table 7: A llelic D iscrim ination PCR R eaction 40X mix

a) A m aster mix or stock solution was m ade using the above ratio o f contents.

b) 10.5ul of the m aster mix solution was then alloqoutted out into each well o f the 96 well optical reaction plate, with 2ul o f genom ic DNA added to every plat except the controls. 10% o f sam ples repeated for quality control.

c) The plate was covered w ith an optical adhesive cover and sealed, prior to being placed in the ABI PRISM 7000 for the PCR reaction and subsequent m easurem ent of fluorescent signalling.

Conditions for the PCR reaction were as out-lined below:

Tim es and Tem peratures Initial Steps Each o f 40 cycles

Denature A nneal/Extend

HOLD CY CLE

10 min 95°C 15 sec 92”C 1 min 60°C Table 8: Therm al C ycler C onditions

Substantial increase in signalling Allele

VIC dye fluorescence only H o m o zy g o sity for A llele 1 6-FA M dye fluorescence only H o m o zy g o sity for A llele 2 B oth fluorescent signals H eterozygotes

Table 9: Dye Signalling and genotype

'V G raph 10.76 3 9.50 - _{— Legend —} 8.00 - 6.50 : 5 .00 I 3.50 = R ♦ 1 /2 ■ No DNA 3.50 4.50 5.00 5.45 0.35 1.00 1.50 2.00 2.50 3.00 A l l e l e I 4.00

Figure 11: Results from TaqM an A llelic D iscrim ination Assay

T here are ty p ically 4 d ifferent clusters o f points in the gen erated results. T h ese points represent the H2O co n tro ls (no am plificatio n )(b lack ), the w ild type (red), the heterozygotes

(green) and the h o m ozygote m utants (blue), (x = u nknow n or u nclear results). (F igure taken fro m A p p lied B iosystem s L td.)

2.9. SNP Selection and Genotyping

2.9.1. TLR7 and 8

Single nu cleo tid e poly m o rp h ism s w ere selected from the N C B I dbS N P database. S electing all SN Ps for w hich frequency data w as available, w e chose from these haplotype tagging SN Ps (htS N P s) to tag all haplotypes presen t in caucasian sam ples at a frequency o f 5% or greater as follow s. Individual g en otypes w ere dow n lo ad ed from d b S N P and haplotye frequencies w ere estim ated co m p u tatio n ally using H IT A G E N E (w w w .h ita g e n e .c o m ) and /or P hase 2.1.1 softw are (S tephens et al 2001). H aplotype tagging SN Ps (tSN Ps) w ere identified using S N P T A G G E R softw are (w w w .w e ll.o x .ac .u k /~ x iav /h a p lo tv p e /) (X iaji et a l 2003). H aplotype tagging S N P s represent the m inim um set o f SN Ps required to id en tify all com m on haplotypes in any given region o f the genom e, for a given population. In addition, SN Ps w ith likely functional co nsequences w ere added to the m in im u m set required to tag haplotype variation. T hus w e selected 4 S N Ps in T L R 7 and 5 SN Ps in T L R 8, (table 10) for genotyping using the A m p liflo u r technique (M yakiskev et a l 2001). T he sequence for the three prim ers required for each assay are outlined in table 12.

SNP Name Rs number Sequence TLR7A Rs 179008 GGACACTGAAGAGAC[A/T]AATTCTTATCCTTTT TLR7B Rs5741880 GTATTGAGTCATATG[G/T]CAGATCCTTTGATCT TLR7C Rs5743780 CAGCCTCATCCAATT[A/G]GATCTGTCTTTCAAT TLR7D Rs5743779 GGGCCTCCCGCCTAG[C/TJTTACAGCTTCTCAGC TLR8A R s3764880 TAGAACAACAGAAAC[A/GJTGGTAAGCCACTTCT TLR8B Rs3747414 TCTGCTAAAAACAAT[A/C]AACAAATCCGCA TLR8C Rs4830808 AATAATGTGGGTCTA[C/T]ACCTCACATTTT TLR8D Rs5744067 CAGTTCCTTGCAG[C/T]TGTTGTTCGACT TLR8E Rs5744077 CCTTCAGTCGTCA[A/G]TGCTGACCTGCA

Table 10: TLR SNP names, Rs numbers and sequences. 2.9.2. DEAD Box SNPs

Single nucleotide polym orphism s were selected from the N CBI dbSN P database. Selecting from SNPs for which frequency data was available, choosing those with a frequency for the rare allele above 20% and those w hich affect putative regulatory or coding regions. Individual genotypes were dow nloaded from dbSNP. Thus we selected 5 SNPs in SKIW 2 and 5 in B A T l, 4 in DDX3 and 2 in RX K/DD X 6, (table JJ) for

genotyping using the A m pliflour technique (M yakiskev e( al 2001). The sequence for the three prim ers required for each assay is outlined in tables 13 & 14.

SNP Name Rs number Sequence

SK Il Rs2280773 GCAGCTGGGA[C/T]GGCTTCCCCTGGAG

SKI2 Rs43899 CCAGAAGGGATAC[C/T]GAGTTGGATTT

SKI3 Rs437179 GAGGCTCCAACA[T/G]ACAGCTAAGGCTT

SKI4 Rs2734331 CACCGAAGGATCTC[C/T]GTGGTCATGAT

SKIS Rs406936 GTTTCAACAT[C/T]TGTCCATGTTCC

BATIA R sI1796 TGGATGTCTTTTA[A/T]GATCAGAAT

BATIB Rs2071595 CTTCCTCCCCCC[C/G]CAACTTTTAGT BATIC Rs2239709 CATTTTGTCCAGG[A/G]TTGTAGTAG BATID Rs2269476 CTTTGGGAGGC[C/T]GAGGCAGGAGGG BATIE Rs2239527 CGAAGGAGGGAAAT[C/G]TGCCTTCACTT RCK l Rs581045 TCCTGTCAAGTA[A/G]TTACTATAGTA RCK2 Rs524590 GAGAGCACATT[A/T]CAGAACAGAGAAGT DDX31 R s6 610546 GAAAATGGGTC[A/G]AACATAGGAACAA DDX32 Rs870208 CATGAAAGCACT[C/T]TTACTTTCTAAAAGT DDX33 Rs 10521420 CATTAGAATGTGA[A/G]ATGGGCTTC DDX34 Rs953114 CTTCACTGGG[A/CJACAAAACTTGGTGTTT

2.10. KBioscience (Amplifluor® assay development)

The TLR7 & 8, SKI2, BATI, RcK and DDX3 SNPs were genotyped

com m ercially by KBiosciences (Hoddesdon, Herts, UK) using A m plifluor® technology.

2.10.1. Introduction to A m plifluor® technology

The A m plifluor® (Chem icon, CA, U.S.A) is a one step genotyping system which analyses previously characterised SNPs, allow ing the sim ultaneous am plification and

detection o f DNA. The universal A m plifluor® is targeted to the SNP of interest by unlabeled Z tailed oligonucleotides. It is the unique sequence o f 21 bases at the Z tail at the 3 ’end of the A m plifluor® SNP prim er which gives it is universality. The Z tail is one o f four parts which make up the Am plifluor® molecule, the others being; a green and red am pliflour, a hairpin structure, and a quencher. The assay utilises two fluorescently labelled A m plifluor® SNP Primers and three unlabelled standard desalted oligonucleotides - two Z-tailed allele-specific primers (one on the 3’ end of both the red and green am pliflour) and a com m on reverse primer (.see Figure 2.7). The SNP primers have a hairpin .structure which is specifically designed to give optimal therm odynamic stability. Unlabeled, allele-.specific oligonucleotides which have the same green or red Z-tail, but on their 5 ’ end, are synthesized. During

com petitive allele specific PCR it is the universal A m pliflour prim ers that generate the fluorescent signal whilst the distinction between the alleles is provided by the unlabeled primers. Incorporation of the A m plifluor® SNP Prim er into an allele-specific amplicon melts its hairpin structure, thus separating the fluorophore from the quencher and generating a fluorescent signal.

SNP G.nr - <\llete G SNP G^rr - A leie T

| Fuiiward aillMltt'-spK::ifK: m a c iia n

f b) R ev erse reactton buildiric th e com plem ent to Ihe ta Is

- A -

F n w a i t l rnjiratnKi wittii ijn iy n rs jil F T p r i n n r j i

F^g'i^erse re a c to T opgnim a th e nairpin s lru d u * ^

Red Green