The amplification of the proto oncogene smoothened

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The aim of this project is to investigate Smoothened (SMO) gene; its functions, structure and it role in Basal cell carcinoma (BCC) using literature review and experimental research. Upon identification of region of interest on the gene because of the implication of this region in some disease state, polymerase chain reaction PCR was used to amplify the region. The PCR product is then separated using Agarose gel electrophoresis, quantified by spectrophotometer and sequenced; occurrence of SNP was then investigated. No single nucleotide polymorphism was observed in the sequencing, however, a deletion of a nucleotide 'C'was observed at base pair 5 and addition of 'A' was also observed at base pair 382.

The aim of this project is to investigate Smoothened (SMO) gene; its functions, structure and it role in Basal cell carcinoma (BCC) using literature review. Upon identification of region of interest on the gene, polymerase chain reaction is use to amplify the gene. The PCR product is then separated using Agarose gel electrophoresis, quantified by spectrophotometer and sequenced; occurrence of SNP is then investigated

1.2) Smoothened gene structure

The genomic DNA containing the human SMO locus was isolated in two non-overlapping λ clones spanning more than 35 kilobases(kb) and sequenced. The human gene is composed of 12 exons within 24kb of genomic DNA (Fig...). Exon1 and 2 contain 5ʹ untranslated sequences, the initiation codon ATG, and the entire signal peptide. The chromosomal location of human SMO was determined by fluorescence in situ hybridization. The λ clone SMO-7was biotinylated and hybridized to metaphase spreads of a normal individual, resulting in specific labelling of the distal long arm of chromosome 7 demostrated that SMO is located 68% of the distance from the centromere to the telomere of chromosome arm 7q, an area corresponding to band 7q31-32.(Xie J. et al.1998)

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Figure 1.1: structure of human chromosome 7 showing the location of SMO gene.( www.ncbi.nlm.nih.gov 4th of July 2010,13.45)

Figure1.2:Human SMO genomic structure and localization. a, Genomic structure of SMO. Exon are represented by filled boxes; grey boxes represent non coding region and black boxes coding regions. The ATG initiation codon and the stopcodon are indicated by lines topped by an arrow an a dot, respectively. The solid line indicates intronic regions for which sequence has been determined; dashed line represent unsequenced gap that has been size by PCR. The CpG island upstream of exon 1 is shown as a cluster of rare restriction-enzyme recognition sites(B,BssHll; S, Sacll; N, Notl;Nr, Narl). The two non-overlapping λ cyclones characterized are shown above the gene structure. B, chromosomal localization of the human SMO gene. Human metaphase cell were hybridized to a digoxigenin-labelled hSMO probe detected by a fluoresceinated antidigoxigenin antibody (green) and to a biotin-labelled centromeric marker of chromosome 7 (D7Z1) revealed by texas-red avidin(red) and counterstained with DAPI. Of a total of 80 metaphase cells analysed, 71 were specifically labelled.(Xie et al. 1998)

1.3) Smoothened protein structure

The protein is a multipass membrane protein and has sequenced length of 787, mass of 86,397 Da (www.uniprot.org/iniprot/A4D1K5, last access on 20/08/10)

Recent research revealed Smo to be an integral membrane protein containing seven membrane-spanning alpha helices, a long N-terminal extracellular peptide, and a C intracellular peptide. Smo is most closely related to the Frizzled (Fz) family of GCPRs, with the Drosophila Smo (dSmo) hepthahelical region showing 31% identity and 52% similarity to that of dFz.

Smo also contains a membrane-intergrated hepthahelical domain, a common feature in all GCPRs. Akin to other GCPRs, many of the known small-molecule regulators of Smo bind to this domain,such as antagonist cyclopamine. Ligand binding to Smo is essential for its regulation because mutations in the heptahelical domain render the protein either inactive or constitutively active. It has also been found that residue in the C-terminal end of the third intracellular loop of GCPRs are often crucial for the G-protein coupling and this could also be true for Smo. For example, an allele of dSmo carrying a mutation in a conserved arginine in this loop exhibits significant decreased activity. Furthermore, several pro-oncogenic Smo mutation(including Smo-M2) within this region(seven transmembrane domain) have been isolated from basal cell carcinomas. Although both invertebrate and vertebrate Smo has diverged significantly between these lineages; the latter does not contain the long cytoplasmic tail present in dSmo, and instead has a short tail with few similarities to its insect counterpart. These differences might account for the disparities between Smo signalling in different organisms. (Ayers et al 2010)

Figure 1.3: structure of the Smoothened protein. Structure of Drosophilia and Human Smo proteins. Numbers represent amino acids. CRD,cystein rich domain. Red stars on the human Smo indicate well-characterized mutation that has been identified in cancer patients.M1,M2 and A2 are some activating mutation isolated from basal cell carcinomas. M1 contains an Arg to Gln changes at amino acid(aa) 562, whereas M2 has a Trp to Leu changeat aa 535. Smo-A2 has Ser533 mutated to one of several non-conserved residues. Smo residue 473 has been implicated in drug(cyclopamine and GDC-0449) binding, and mutation of this residue has been found in GDC-0449 resistant medulloblastomas.(Ayers et al 2010)

1.3.1) Protein function

Smo is one of the proto-oncogene that plays an important role in the human development. It's a vital part of the Hegdehog signalling pathway, it's present if not restricted by a Patched gene (PTCH1) a receptor of sonic hedgehog can lead to carcinogenesis.

The family of hedgehog protein includes the secreted protein SHH, first identified as an important regulator of segment polarity in Drosophilia. The amino-terminal peptide of the autocatalytically cleaved SHH protein triggers pathway activation by binding to the transmembrane protein PTCH, which relieves its inhibition of the SMO. Thus, depression of SMO result in activation of signalling cascade via the GLI transcription factors and lead to the transcription of numerous downstream target genes including members of the transforming growth factor β and WNT families, as well as PTCH.Abnormalities in the SHH signalling pathway have been found in various developmental pathologies and also in neoplasms

(Couve privat et al 2002)

http://www5.appliedbiosystems.com/tools/pathway/loadImage.php?pos=bl&im=images/Hedgehog%20Signaling%20in%20Mammals.jpg

Figure 1.4 HEDGEHOG SIGNALLING PATHWAY

1.3.2) Phosphorylation of Smo

Activation of Smoothened gene is believe to be via it phosphorylation.Hh protein binding to the transporter-like receptor Patched (Ptc) releases an inhibitory effect of Ptc on Smoothened . In Drosophilia (which is a homologue SMO gene to SMOH in human), activated Smo accumulate at the plasma membrane and signals pathway activation by recruiting a complex of intracellular pathway components,including the Kinesin-like molecules Costal-2 (Cos2),the putative serine/threonine protein Kinase Fused(Fu), and Cubitus interruptus (Ci), a Zinc finger transcription factor that is a major transcriptional effector of the pathway. In the abscence of Hh stimulation, Cos 2 tethers Ci in the cytoplasm and promote it cleavageto a repressor form, CiR, whereas the supresspr of Fused[Su(Fu)] protein associate with and negatively regulate the fraction of full-length Ci that is not associated with Cos2/Fu/Ci complex, at least in part by influencing its subcellular localization. Upon Hh-induced activation , the Cos2/Fu/Ci complex is recruited to the membrane through Cos2 association with the Smo cytoplasmic tail, and Su(Fu) is inactivated through the activity of Fu. This event lead to decreased Ci cleavage and loss of CiR repressor, accumulation and nuclear translocation of Ci,and expression of Hh pathway transcription target

Hh pathway activation is marked by decreased phoshorylation of Ci and increased phoshorylation of Smo,Cos2, Fu and Su(Fu), which can be observed withing 10-15 min of stimulation. Phosphorylation of Ci maintains pathway quiescence by promoting cleavage of Ci to CiR, and at least three kinases, cAMP-dependent protein kinase (PKA), casein KINASE 1α (CK1α), and glycogen synthase Kinase 3β (GSK3 β),likely exert their negative effect on pathway activity through Ci phosphorylation.( Zhang et al.2004)

Figure 1.5: Predicted and detected phosphorylation in SMO. A schematic diagram of SMO is shown alongside specific residues within intracellular loops two and a portion of the C-terminal cytoplasmic tail. Identified phosphoserin-threonine residue is endogenous SMO purified from HhN-stimulated Drosophila S2 cell marked with asterisks. Phosphopeptides were isolated from distinct region as designated (1-Vlll). Residue within the concesus kinase recognition motifs shown for PKA (red) and CK1 or GSK3, depending on the priming phosphoresidue is shown in green. Designation of CK1 and GSK3 target residues assuming that all possible priming phosphorylationhas occur (Zhang et al 2004)

1.4) Mutation of Smoothened gene

Mutation of SMO had been identify as a cause of some cancer. In cancer such as Basal Cell Carcinoma(BCC),SMO gene was screen for activating mutation using heteroduplex and single strand conformation polymorphism(SSCP) analysis. Variant band were detected in exon 9 and 10 of some of the BCC sample analysed. Seqeunce analysis of exon 9 heteroduplex variant band identified the identical heterozygous mutation in the analysed tumor samples,a G-to-T transversion at base pair 1,604,changing codon 535 from Trp to Leu. The screening of the blood DNA sample of the patients from which the mutant SMO samples were taken, did not contain this sequence alteration, indicating that the mutation in the tumour arose somatically. The Trp residue at codon 535 is conserved among all analysed SMO protein sequences,is located in the seventh transmembrane domain and correspond to a conserved Tyr residue in most G-protein-coupled receptors. In particular, this residue in the α1B -adrenergic receptors is thought to be involved in the formation of a polar pocket that keep the receptor in a latent state. Distruption of this pocket by mutagenesis lead to constitutive signalling by the α1B -adrenergic receptors . The mutant Trp residue in SMO could cause unrestrained SMO signalling would be by interference with the binding of SMO by PTCH, and hence a loss of the normal PTCH inhibition of SMO function.( Xie et al. 1998)

Figure 1.6 schematic representation of the location of mutation on the SMO gene. The SMO protein with the seven transmembrane domains is presented. The numbers refer to mutated codons with corresponding exons in parenthesis. Black arrowheads denote point mutation in BCC from XP patient, and gray arrowheads denote point mutation in sporadic BCC.(Couve-Privat et al. 2002)

Figure 1.7 Skin of SMO-M2 transgenic mice display the features of basal cell carcinoma. a,b, Haematoxylic-eosin-stained sections of SMO-M2 (a) and WT (b) transgenic E18 mouse skin. SB, stratum basale; SS,statum spinosum; SC,stratum corneum;SG,stratum granusolum;PBC,polisading basal cell of epithelial pegs (EP);HF, hair follicles. c, d, Over-expression of SMO detected in the epithelium of transgenic mice using antibody directed against a tag inserted at the N terminus of the construct. Expression was limited to the basal cell layer SMO -M2 mice (c) and not detected in wild type mice (d). e, f, Accumulation of PTCH RNA, detected by in situ hybridization using a PTCH riboprobe in basal cells of mice overexpression SMO-M2(e) compare to wild-type mice(f)( Xie et al.1998)

1.4.1 Role of SMO in BCC cancer

The development of basal cell carcinoma is associated with constitutive activation of sonic hedgehog signaling. Normally, ligand-dependent signaling by Hedgehog (Hh) homologs proceeds through binding to the Patched receptor. This binding relieves the Patched-mediated inhibition of signaling through the Smoothened (SMOH) gene product. This signaling ultimately results in the dissociation of the Gli1 transcription factor from an inhibitory complex in the cytoplasm, its subsequent translocation to the nucleus, and activation of target gene expression. The mutations in SMOH, PTCH1, and SHH in BCCs result in continuous activation of target genes. At a cellular level, sonic hedgehog signaling promotes cell proliferation. Mutations in TP53 are also found with high frequency (>50%) in sporadic BCC. (Kyoto encyclopaedia of genes and genomes, 2009)

1.5 possible treatment of smoothened implicated diseases

Cyclopamine possible treatment of Smoothened implicated cancer disease

The steroidal alkaloid cyclopam has both teratogenic (able to distrupt growth and development of embryo) and antitumor activities arising from it ability to specifically inhibit cellular response to vertebrate hedgehog signalling. Recent study shows using photoaffinity and fluorescent derivatives ,that this inhibitory effect is mediated by direct binding of cyclopamine to the heptahelical bundle of Smoothened(Smo). Cyclopamine also can reverse the retention of partially misfolded Smo in the endoplasmic reticulum,presumably through binding-mediated effects on protein conformation. Cyclopamine has shown some promise in the treatment of medullablastoma tumors cause by inappropriate Hh pathway activation. How cyclopamine specifically inhibit Hh pathway activation is unclear,but it appears to interfere with the initial events of vertebrate Hh signal reception,which involve the multipass trasmembrane (TM) protein Patch Smoothened. Cyclopamine appears to interfere with the Hh signal by influencing Smo function,as it antagonizes Hh pathway activity in a Ptch-independent manner and exhibit attenuating potency toward an oncogenic,constitutively active form of Smo.(Chen et al. 2010)

GDC-0449 possible treatment of smoothened implicated cancer

GDC-0449 is a novel smoothened inhibitor which was discovered by high throughtput screening of small molecules and subsequent optimization( Von hoff et al, 2009). It's an orally bioavailable molecule that has a higher potency and pharmaceutical properties than cyclopamine. Previous research using mouse model shows GDC-0449 has antitumor activity that through inhibition of hedegehog pathway( Rudin et al,2009)

1.6 Polymerase Chain Reaction (PCR)

The polymerase chain reaction is a test tube system for DNA replication that allows selective amplification of a target DNA sequence to several million-fold in just a few hours. In the PCR reaction a thermolabile DNA polymerase (Taq) is used for the amplification of a small fraction of the genome.

2.0 CHAPTER TWO MATERIALS AND METHODS

2.1 MATERIALS AND APPARATUS

The materials and apparatus used for this experiments are:

Human placental DNA (1µg/ml)

Enzyme mix made up of:

Taq polymerase

Reaction buffer

Deoxynucleotide (dNTPs)

Sterile deionized water (SDW)

MgCl2

GelRed

Agarose pwder

Forward and reverse Primers

Step ladder

Lamder DNA

PCR tubes

Pipettes

Balance

Conical flask

Spectrophotometer

Microwave oven

PCR machine

Comb

2.2) Methods

2.2.1 Bioinformatic methods

The first step of this project involves the research of Smoothened gene in the bioinformatic database. Database such as the American National Centre for Biotechnology Information (NCBI),gives alot of information such as DNA structure, domain, gene expression, location of the gene and the protein information (www.ncbi.nlm.nih.gov ). The UCSC database (http://genome.ucsc.edu/) was used to identify the sequence showing all the intron and exon present in the gene. Exons of interest was chosen from this sequence based on previous research work that linked this exon to a particular disease (Xie et al. 1998), in this project; exon 9. Once the above procedure was done Primer3 (http://frodo.wi.mit.edu/primer3/) software was used to run the PCR (polymerase chain reaction) and the Insilico PCR . As the Insilico PCR was successful, primer are then ordered from the Sigma life science (www.sigma.com/oligos ) and the laboratory work then start.

2.2.2 PCR METHOD

As earlier discussed the PCR is used to amplified the region of interest of the Smoothened gene. To do this much care was taking to avoid contamination such as lose DNA from me and the environment, this was achieved by wearing of gloves at all time, closing all the sample containers when not in use, each eppendorf tube should be kept separately, pipette tip must not be reuse and all reagent was conserved in ice to avoid denaturation.

The first Step was making solution of the forward and reverse primer purchased from SIGMA genosys. The dehydrated primers contains 63.6nmol of the forward primer and 48.0nmol of the reverse primer respectively. Each primer was diluted with standard deionized water (SDW) to make 100µM each, this is done by adding 636µl of SDW to the forward primer and 480µ1 of SDW to the reverse primer. 10µ1 of each primer was taken from the 100µl solutions and was pipette into a separate eppendorf tube and was make up 100µl with 90µl of SDW to give a working stock of 10µM in each tube. The stock is the conserved in the freezer at -20oc

PCR EXPERIMENT 1

All the Eppendorf tubes used for this experiment were kept on ice until they are ready for insertion into the thermocycler.

A master mix was prepared three times the volume needed to prevent shortage that may arise from pipetting error. Two replicate samples was perform and the third being the control. The component were added and briefly spined in a microfuge as detailed below:

PCR mixture for an individual tube/reaction: 3.5x

Forward primer (10µM) =1µl 3.5µl

Reverse primer (10µM) =1µl 3.5µl

Enzyme mix =10µl 35µl

Sterile deionized water =7µl ˜25µl

.

Total Master mix =19µl

Human placental DNA =1µl

Total =20µl

For the control 1µ1 of SDW instead of 1µl of human placental DNA was added to 19µl of the Master mix.

The samples were inserted into the PCR machine and start the PCR programme in the following manner

PCR programme

Initial denaturation 94oC 5 minutes- 1cycle

Denaturation 94 oC 1minutes} 30 cycles

Annealing 59 oC 30 seconds } 30 cycles

Extension 72 oC 1minute} 30 cycles

Final extension 72 oC 5minutes - cycle

Annealing temperature = Tm of the forward and reverse primer minus 5 oC i.e( 65-5) oC

The overall programme last for 1hr:41min. The PCR product (amplified gene) was then separated using gel electrophoresis

2.2.3 Gel electrophoresis

Gel electrophoresis was used to separate the amplified DNA band. 1% w/v agarose gel was prepare by accurately weighing out 1g of agarose powder and making up to 100ml with TBE( Tri-HCL, boric acid and EDTA) in a conical flask. The solution was heated in a microwave for about one minute until the solute dissolve completely. When the solution becomes lukewarm, about 10µl red gel was added. The solution stirred and poured into the casting tray, a comb was place into it to form the sample well and the whole set up was left for about an hour to set.

When the PCR programme finish running, the product is taking out and about 10µl of samples were taking for the gel electrophoresis. The remaining 10µl was used for DNA quantification and sequencing. The gel tank was filled with TBE not exceeding the tank limit mark, then 10µl of the step ladder was loaded into the first well. 10µl of the control and the PCR products were also loaded into the other well on the gel. The gel was run at a constant voltage of 70V for about 1:30 hours when the coloured sample, step ladder and control migrated about 2/3 of the gel. At this point the current turned off and the gel examine by a UV transluminator to detect the extent of migration of the sample with respect to the step ladder.

2.2.4 DNA Quantification

The DNA quantification is necessary because I needed to know the quantity of amplified DNA I was sending to COGENIC for sequencing. To do this UV spectrophotometer was used following the steps below:

To Zero the spectrophotometer, 995µl of SDW and 5µl of red enzyme mix was added to a disposable cuvette (UV range) and carefully mixed using a pipettor with 1ml(blue) tip. The cuvette was place in the spectrophotometer(at 250nm) and set to read zero.

A standard reading was also taking using 994µl of SDW, 5µl of red enzyme mix and 1µl(458ng) of a known concentration of phage lamda (λ) DNA. The solution was mixed as described above and the reading was taking at 260nm.

Finally, the amplified DNA was measured: carefully mixing 990µl of SDW with 10µl of amplified DNA and the reading was taking.

To calculate the concentration of the nominal DNA, the following formula was used:

Nominal DNA concentration(µg/ml)=A260 x 50 x dilution factor

A260= Absorbance reading at 260nm wavelength

Dilution factor = in this case for λ DNA is 1000 and 100 in the case of amplified DNA.

The whole spectrophometry reading usually gives an overestimated values, so the DNA concentration had to be adjusted to a realistic value. To do this the following procedure was adopted:

Since the λ DNA was used as a standard i.e. the quantity was known and in this case was 458ng/µl. The concentration of the λ DNA calculated from the absorbance reading was divided by the true value(458ng/µl).

The next step involves preparation of the forward and reverse primer for sequencing. 20µl of 0.8pmol/µl of each primers is required for the sequencing from the original 100µM stock solution. This is done by using the following calculation:

Preparation primer solution

The information provided by SIGMA( primer designer) says '63.6nmol of the primer should be diluted with 636µl of SDW to make 100µM. So if 63.6nmol is contained in 636µl of SDW

Which is the same as 636µl of SDW contain 63600pmol of the primer

So 1µl of SDW will contain 63600/636 =100pmol/µl

Using the formula

C1V1 =C2V2

100pmol/µl x V1 =0.8pmol/µl x 20µl

=0.16µl

But because I don't have a pipettor that has 0.16µl capacity I made my V2=250µl instead

So the calculation goes thus:

100pmol/µl x V1 =0.8pmol/µl x 250µl

=2µl

Therefore 2µl of the forward primer was diluted with 248µl of SDW to give 0.8pmol/µl. 20µl was then taking from this solution and sent for the sequencing.

CHAPTER 3 RESULT AND DISCUSSION

3.1 Primer design

PCR amplification begins with the design of the part of gene that is of interest. This is the step that really determines the outcome of the whole project. Therefore, care must be taking in choosing a particular regions of interest for analysis of mutation implicated in a certain disease. The primers are design specifically for the region to amplify.

The human SMO gene contain twelve exons and all twelve code for the gene. In this project exon 9 was amplified based on reported mutation in this region implicated in basal cell carcinoma( Xie et al. 1998)

10 20 30 40 50 60

MAAARPARGP ELPLLGLLLL LLLGDPGRGA ASSGNATGPG PRSAGGSARR SAAVTGPPPP

70 80 90 100 110 120

LSHCGRAAPC EPLRYNVCLG SVLPYGATST LLAGDSDSQE EAHGKLVLWS GLRNAPRCWA

130 140 150 160 170 180

VIQPLLCAVY MPKCENDRVE LPSRTLCQAT RGPCAIVERE RGWPDFLRCT PDRFPEGCTN

190 200 210 220 230 240

EVQNIKFNSS GQCEVPLVRT DNPKSWYEDV EGCGIQCQNP LFTEAEHQDM HSYIAAFGAV

250 260 270 280 290 300

TGLCTLFTLA TFVADWRNSN RYPAVILFYV NACFFVGSIG WLAQFMDGAR REIVCRADGT

310 320 330 340 350 360

MRLGEPTSNE TLSCVIIFVI VYYALMAGVV WFVVLTYAWH TSFKALGTTY QPLSGKTSYF

370 380 390 400 410 420

HLLTWSLPFV LTVAILAVAQ VDGDSVSGIC FVGYKNYRYR AGFVLAPIGL VLIVGGYFLI

430 440 450 460 470 480

RGVMTLFSIK SNHPGLLSEK AASKINETML RLGIFGFLAF GFVLITFSCH FYDFFNQAEW

490 500 510 520 530 540

ERSFRDYVLC QANVTIGLPT KQPIPDCEIK NRPSLLVEKI NLFAMFGTGI AMSTWVWTKA

550 560 570 580 590 600

TLLIWRRTWC RLTGQSDDEP KRIKKSKMIA KAFSKRHELL QNPGQELSFS MHTVSHDGPV

610 620 630 640 650 660

AGLAFDLNEP SADVSSAWAQ HVTKMVARRG AILPQDISVT PVATPVPPEE QANLWLVEAE

670 680 690 700 710 720

ISPELQKRLG RKKKRRKRKK EVCPLAPPPE LHPPAPAPST IPRLPQLPRQ KCLVAAGAWG

730 740 750 760 770 780

AGDSCRQGAW TLVSNPFCPE PSPPQDPFLP SAPAPVAWAH GRRQGLGPIH SRTNLMDTEL

MDADSDF

Figure 3.1 Protein sequence of SMO. The protein contain 787 Amino acid, a mass of 86,397 and a genomic size of 24,671 (http://www.uniprot.org/uniprot/Q99835#section_seq 10 July 2010, 15.00)

The primers are designed using UCSC to first acquire the genomic sequence of the SMO gene and selecting the appropriate exon, in this case exon 9.

aagaaaggaaagcctcacctgtctacgttccctcactgtagATGTCAGGC

CAATGTGACCATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGA

TCAAGAATCGCCCGAGCCTTCTGGTGGAGAAGATCAACCTGTTTGCCATG

TTTGGAACTGGCATCGCCATGAGCACCTGGGTCTGGACCAAGGCCACGCT

GCTCATCTGGAGGCGTACCTGGTGCAGgtgggcatggcagccagcccctc

ctgccctgcccgcctcaccctcagccttgggaccccatctttaggttttg

tcgggtcctgcctctagcacagccttggtcagtggttcaccgctgccccc

tggtggcaccttctgtccttgggtggcctgatgcctgggcctgggcctgg

gccaagggcagagccagctgccatcaccttttaagagcggagtgattgga

gggctgggcacacaggagctctgcattctggccccacttctttgcagaga

aggcctctactcctgagtccttgaaggacttgaggcccttgggagcctcc

Figure 3.2 genomic sequence of exon 9 of Smo gene(www.ucsc.edu/ 20th August 2010,16.31)

The selected region of the gene above was used to design the primers using primer3 software using 501-1000 bp product size. The result is display below

No mispriming library specified

Using 1-based sequence positions

OLIGO start len tm gc% any 3' seq

LEFT PRIMER 12 20 59.73 60.00 4.00 0.00 gcctcacctgtctacgttcc

RIGHT PRIMER 540 20 61.12 50.00 7.00 3.00 aagggcctcaagtccttcaa

SEQUENCE SIZE: 550

INCLUDED REGION SIZE: 550

PRODUCT SIZE: 529, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00

1 aagaaaggaaagcctcacctgtctacgttccctcactgtagATGTCAGGCCAATGTGACC

>>>>>>>>>>>>>>>>>>>>

61 ATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGATCAAGAATCGCCCGAGCCTT

121 CTGGTGGAGAAGATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCATGAGCACCTGG

181 GTCTGGACCAAGGCCACGCTGCTCATCTGGAGGCGTACCTGGTGCAGgtgggcatggcag

241 ccagcccctcctgccctgcccgcctcaccctcagccttgggaccccatctttaggttttg

301 tcgggtcctgcctctagcacagccttggtcagtggttcaccgctgccccctggtggcacc

361 ttctgtccttgggtggcctgatgcctgggcctgggcctgggccaagggcagagccagctg

421 ccatcaccttttaagagcggagtgattggagggctgggcacacaggagctctgcattctg

481 gccccacttctttgcagagaaggcctctactcctgagtccttgaaggacttgaggccctt

<<<<<<<<<<<<<<<<<<<<

541 gggagcctcc

KEYS (in order of precedence):

>>>>>> left primer

<<<<<< right primer

Figure3.3: sequencing and annealing position of two primer using primer3( http://frodo.wi.mit.edu/primer3/ 14th July 2010, 12.00)

Insilico PCR run

chr7:128850244+128850760 517bp CCCATCCCTGACTGTGAGAT TCCCATCAAATCTTGGGAAG

CCCATCCCTGACTGTGAGATcaagaatcgcccgagccttctggtggagaa

gatcaacctgtttgccatgtttggaactggcatcgccatgagcacctggg

tctggaccaaggccacgctgctcatctggaggcgtacctggtgcaggtgg

gcatggcagccagcccctcctgccctgcccgcctcaccctcagccttggg

accccatctttaggttttgtcgggtcctgcctctagcacagccttggtca

gtggttcaccgctgccccctggtggcaccttctgtccttgggtggcctga

tgcctgggcctgggcctgggccaagggcagagccagctgccatcaccttt

taagagcggagtgattggagggctgggcacacaggagctctgcattctgg

ccccacttctttgcagagaaggcctctactcctgagtccttgaaggactt

gaggcccttgggagcctccttctctggaaagaatggcatcgctggccCTT

CCCAAGATTTGATGGGA

Figure3.4 sequence of the insilico PCR(http://genome.ucsc.edu/ 14th July 2010,13.00)

3.2 PCR amplification

The amplification of the PCR region was carried out several times under different condition inorder to find the most suitable annealing temperature. The annealing temperature provided by SIGMA for the foreward and reverse primers are 63.7oC and64.9oC respectively. The operational annealing temperature should be 5oC minus the two temperatures above, thus, the annealing temperature becomes ˜59oC .

The first experiment was carried out as described in the materials and method and viewed in the transiluminator. The result shows good bands that correspond to base pair of the gene (500 bp). However, there was evidence of some contamination

S 1 2 3

↓ ↓ ↓ ↓D:\scan 1 at 59 C.jpg

Figure 3.5 PCR carried out at 59oC. The band showing the step ladder S, channel l is the control, channel 2 and 3 are the amplified PCR gene

The second experiment was carried out using annealing temperature of 59oC. However, the gene was diluted in the ratio of 4:4 i.e.4µl of Template DNA diluted with 4µl of SDW. The result was encouraging showing good bands at about 500bp when measured using the step ladder as a standard. The result is display below:

S 1 2 3

↓ ↓ ↓ ↓

D:\SCAN at 59 c ( 1uL of DNA from 4uL of DNA +4uL of H2O.jpg

Figure 3.6. PCR amplification using a diluted template gene in the ratio of 4µl to 4µl of SDW at 59oC. Channel S represent the step ladder, channel 1 is the control, channels 2 and 3 is the amplified PCR sample.

Subsequent experiment was performed using the same annealing temperature as the previous experiment but the gene wasn't diluted. The amplified gene was re PCR to get enough quantity for sequencing of the gene. The results was shown below:

S 1 2 3 4 5 6 7

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

D:\20-06-10 @ 59c.JPG

Figure 3.7 re PCR1 amplification carried out at 59oC. S represent the step ladder. Channel 1 is the control. Channel 2-7 are six replicate of amplified gene.

S 1 2 3 4 5 6 7

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

D:\26-07-10 PCR 59oC.jpg

Figure 3.8 re PCR1 amplification carried out at 59oC. S represent the step ladder. Channel 1 is the control. Channel 2-7 are six replicate of amplified gene.

3.3) Sequencing

Sequence obtain for the forward primer

GTGACATCGGGCTGCCCACCAAGCAGCCCATCCCTGAC

TGTGAGATCAAGAATCGCCCGAGCCTTCTGGTGGAGAA

GATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCA

TGAGCACCTGGGTCTGGACCAAGGCCACGCTGCTCATC

TGGAGGCGTACCTGGTGCAGGTGGGCATGGCAGCCAGC

CCCTCCTGCCCTGCCCGCCTCACCCTCAGCCTTGGGAC

CCCATCTTTAGGTTTTGTCGGGTCCTGCCTCTAGCACA

GCCTTGGTCAGTGGTTCACCGCTGCCCCCTGGTGGCAC

CTTCTGTCCTTGGGTGGCCTGATGCCTGGGCCTGGGCC

TGGGCCAAGGGCAGAGCCAGCTGCCATCACCTTTTAAG

AGCGGAGTGATTGGAGGGCTGGGCACACAGGAGCTCTG

CATTCTGGCCCCACTTCTTTGCAGAGAAGGCCTCTACT

CCTGAGTCCTTGAAGGACTTGAAGGCCCTTAA

Figure 3.9: Sequence of the amplified gene using the forward primer

Sequence obtained with the reverse primer

CAAGAAGTGGGGCCAGAATGCAGAGCTCCTGTGTGC

CCAGCCCTCCAATCACTCCGCTTCTTAAAAGGTGA

TGGCAGCTGGCTCTGCCCTTGGCCCAGGCCCAGGC

CCAGGCATCAGGCCACCCAAGGACAGAAGGTGCCA

CCAGGGGGCAGCGGTGAACCACTGACCAAGGCTGT

GCTAGAGGCAGGACCCGACAAAACCTAAAGATGGG

GTCCCAAGGCTGAGGGTGAGGCGGGCAGGGCAGGA

GGGGCTGGCTGCCATGCCCACCTGCACCAGGTACG

CCTCCAGATGAGCAGCGTGGCCTTGGTCCAGACCC

AGGTGCTCATGGCGATGCCAGTTCCAAACATGGCA

AACAGGTTGATCTTCTCCACCAGAAGGCTCGGGCG

ATTCTTGATCTCACAGTCAGGGATGGGCTGCTTGG

TGGGCAGCCCGATGGTCACATTGGCCTGACATCTA

CAGTGAGGGAACGTAGACGGGTGAGGCA

Figure3.10: Sequence of the amplified gene using the reverse primer

The reverse primer was complemented using the website 'cellbiol' following this link (http://www.cellbiol.com/scripts/complement/reverse_complement_sequence.html 24th August 2010). This is necessary inorder to join the forward primer to the reverse

Sequence obtained for the reverse compliment

TGCCTCACCCGTCTACGTTCCCTCACTGTAGATGTCAG

GCCAATGTGACCATCGGGCTGCCCACCAAGCAGCCCAT

CCCTGACTGTGAGATCAAGAATCGCCCGAGCCTTCTGG

TGGAGAAGATCAACCTGTTTGCCATGTTTGGAACTGGC

ATCGCCATGAGCACCTGGGTCTGGACCAAGGCCACGCT

GCTCATCTGGAGGCGTACCTGGTGCAGGTGGGCATGGC

AGCCAGCCCCTCCTGCCCTGCCCGCCTCACCCTCAGCC

TTGGGACCCCATCTTTAGGTTTTGTCGGGTCCTGCCTC

TAGCACAGCCTTGGTCAGTGGTTCACCGCTGCCCCCTG

GTGGCACCTTCTGTCCTTGGGTGGCCTGATGCCTGGGC

CTGGGCCTGGGCCAAGGGCAGAGCCAGCTGCCATCACC

TTTTAAGAAGCGGAGTGATTGGAGGGCTGGGCACACAG

GAGCTCTGCATTCTGGCCCCACTTCTTG

Figure 3.11: Sequence of the reverse complement obtained from (http://www.cellbiol.com/scripts/complement/reverse_complement_sequence.html, 24th August 2010, 09.00)

The sequence obtained from the left and right primer are clearly from the same amplicon, however, they do not match completely. This may be due to the fact sequencing reaction are only accurate for a few hundred base pairs. So, to join the forward and the reverse primer, a set of nucleotide from the forward primer were search for in the reverse complement. At the point of matching i.e. overlapping i join the primer. The sequence below shows the joined primer:

GTGACATCGGGCTGCCCACCAAGCAGCCCATCCCTGAC

TGTGAGATCAAGAATCGCCCGAGCCTTCTGGTGGAGAA

GATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCA

TGAGCACCTGGGTCTGGACCAAGGCCACGCTGCTCATC

TGGAGGCGTACCTGGTGCAGGTGGGCATGGCAGCCAGC

CCCTCCTGCCCTGCCCGCCTCACCCTCAGCCTTGGGAC

CCCATCTTTAGGTTTTGTCGGGTCCTGCCTC

TAGCACAGCCTTGGTCAGTGGTTCACCGCTGCCCCCTG

GTGGCACCTTCTGTCCTTGGGTGGCCTGATGCCTGGGC

CTGGGCCTGGGCCAAGGGCAGAGCCAGCTGCCATCACC

TTTTAAGAAGCGGAGTGATTGGAGGGCTGGGCACACAG

GAGCTCTGCATTCTGGCCCCACTTCTTG

Figure3.12: Sequence obtained from combination of the forward and reverse primers. The red highlight indicate where the sequence were overlapped.

The joined sequence was blasted using pairwise BLAST following this link (http://blast.ncbi.nlm.nih.gov/Blast.cgi 25th August 2010, 14:29)

Score = 316 bits (171), Expect = 5e-84

Identities = 174/175 (99%), Gaps = 1/175 (0%)

Strand=Plus/Plus

Query 1 GTGA-CATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGATCAAGAATCGCCCG 59

|||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1760 GTGACCATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGATCAAGAATCGCCCG 1819

Query 60 AGCCTTCTGGTGGAGAAGATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCATGAGC 119

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1820 AGCCTTCTGGTGGAGAAGATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCATGAGC 1879

Query 120 ACCTGGGTCTGGACCAAGGCCACGCTGCTCATCTGGAGGCGTACCTGGTGCAGGT 174

|||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1880 ACCTGGGTCTGGACCAAGGCCACGCTGCTCATCTGGAGGCGTACCTGGTGCAGGT 1934

>ref|NT_007933.15| Download subject sequence NT_007933 spanning the HSP Homo sapiens chromosome 7 genomic contig, GRCh37 reference primary

assembly

Length=77412220

Features in this part of subject sequence:

smoothened

Score = 798 bits (432), Expect = 0.0

Identities = 437/439 (99%), Gaps = 2/439 (0%)

Strand=Plus/Plus

Query 1 GTGA-CATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGATCAAGAATCGCCCG 59

|||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 66883060 GTGACCATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGATCAAGAATCGCCCG 66883119

Query 60 AGCCTTCTGGTGGAGAAGATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCATGAGC 119

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 66883120 AGCCTTCTGGTGGAGAAGATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCATGAGC 66883179

Query 120 ACCTGGGTCTGGACCAAGGCCACGCTGCTCATCTGGAGGCGTACCTGGTGCAGGTGGGCA 179

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 66883180 ACCTGGGTCTGGACCAAGGCCACGCTGCTCATCTGGAGGCGTACCTGGTGCAGGTGGGCA 66883239

Query 180 TGGCAGCCAGCCCCTCCTGCCCTGCCCGCCTCACCCTCAGCCTTGGGACCCCATCTTTAG 239

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 66883240 TGGCAGCCAGCCCCTCCTGCCCTGCCCGCCTCACCCTCAGCCTTGGGACCCCATCTTTAG 66883299

Query 240 GTTTTGTCGGGTCCTGCCTCTAGCACAGCCTTGGTCAGTGGTTCACCGCTGCCCCCTGGT 299

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 66883300 GTTTTGTCGGGTCCTGCCTCTAGCACAGCCTTGGTCAGTGGTTCACCGCTGCCCCCTGGT 66883359

Query 300 GGCACCTTCTGTCCTTGGGTGGCCTGATGCCTGGGCCTGGGCCTGGGCCAAGGGCAGAGC 359

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 66883360 GGCACCTTCTGTCCTTGGGTGGCCTGATGCCTGGGCCTGGGCCTGGGCCAAGGGCAGAGC 66883419

Query 360 CAGCTGCCATCACCTTTTAAGAAGCGGAGTGATTGGAGGGCTGGGCACACAGGAGCTCTG 419

|||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||

Sbjct 66883420 CAGCTGCCATCACCTTTTAAGA-GCGGAGTGATTGGAGGGCTGGGCACACAGGAGCTCTG 66883478

Query 420 CATTCTGGCCCCACTTCTT 438

|||||||||||||||||||

Sbjct 66883479 CATTCTGGCCCCACTTCTT 66883497

>ref|NW_001839071.2| Download subject sequence NW_001839071 spanning the HSP Homo sapiens chromosome 7 genomic contig, alternate assembly

(based on HuRef), whole genome shotgun sequence

Length=28934855

Features in this part of subject sequence:

smoothened

Score = 798 bits (432), Expect = 0.0

Identities = 437/439 (99%), Gaps = 2/439 (0%)

Strand=Plus/Minus

Query 1 GTGA-CATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGATCAAGAATCGCCCG 59

|||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 2443056 GTGACCATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGATCAAGAATCGCCCG 2442997

Query 60 AGCCTTCTGGTGGAGAAGATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCATGAGC 119

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 2442996 AGCCTTCTGGTGGAGAAGATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCATGAGC 2442937

Query 120 ACCTGGGTCTGGACCAAGGCCACGCTGCTCATCTGGAGGCGTACCTGGTGCAGGTGGGCA 179

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 2442936 ACCTGGGTCTGGACCAAGGCCACGCTGCTCATCTGGAGGCGTACCTGGTGCAGGTGGGCA 2442877

Query 180 TGGCAGCCAGCCCCTCCTGCCCTGCCCGCCTCACCCTCAGCCTTGGGACCCCATCTTTAG 239

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 2442876 TGGCAGCCAGCCCCTCCTGCCCTGCCCGCCTCACCCTCAGCCTTGGGACCCCATCTTTAG 2442817

Query 240 GTTTTGTCGGGTCCTGCCTCTAGCACAGCCTTGGTCAGTGGTTCACCGCTGCCCCCTGGT 299

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 2442816 GTTTTGTCGGGTCCTGCCTCTAGCACAGCCTTGGTCAGTGGTTCACCGCTGCCCCCTGGT 2442757

Query 300 GGCACCTTCTGTCCTTGGGTGGCCTGATGCCTGGGCCTGGGCCTGGGCCAAGGGCAGAGC 359

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 2442756 GGCACCTTCTGTCCTTGGGTGGCCTGATGCCTGGGCCTGGGCCTGGGCCAAGGGCAGAGC 2442697

Query 360 CAGCTGCCATCACCTTTTAAGAAGCGGAGTGATTGGAGGGCTGGGCACACAGGAGCTCTG 419

|||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||

Sbjct 2442696 CAGCTGCCATCACCTTTTAAGA-GCGGAGTGATTGGAGGGCTGGGCACACAGGAGCTCTG 2442638

Query 420 CATTCTGGCCCCACTTCTT 438

|||||||||||||||||||

Sbjct 2442637 CATTCTGGCCCCACTTCTT 2442619

Figure 3.13: BLAST performed on the amplified region of SMO gene using the sequence obtained from overlapping of both primer(figure 3.12). The deletion is highlighted in green colour,addition with blue and single polymorphism in red. ( (http://blast.ncbi.nlm.nih.gov/Blast.cgi 25th August 2010, 16.00).

The location of the deletion and addition was searched for in the genomic sequence of the SMO gene and it was found to occur on exon 9. The deletion occurs in the base pair number 5 of the query. The deletion can be due to errors in the sequencing process, however if the deletion is real it can definitely cause a problem such as cancer because it occurs in exon 9 which is one the coding exons of the gene. In the other hand the addition of nucleotide occurs at base pair 382 of the query which when located on genomic sequence occurs in the intron region. The addition does not have any significant effect because it is located in the intron region.

aagaaaggaaagcctcacctgtctacgttccctcactgtagATGTCAGGC

CAATGTGACCATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGA

TCAAGAATCGCCCGAGCCTTCTGGTGGAGAAGATCAACCTGTTTGCCATG

TTTGGAACTGGCATCGCCATGAGCACCTGGGTCTGGACCAAGGCCACGCT

GCTCATCTGGAGGCGTACCTGGTGCAGgtgggcatggcagccagcccctc

ctgccctgcccgcctcaccctcagccttgggaccccatctttaggttttg

tcgggtcctgcctctagcacagccttggtcagtggttcaccgctgccccc

tggtggcaccttctgtccttgggtggcctgatgcctgggcctgggcctgg

gccaagggcagagccagctgccatcaccttttaagaagcggagtgattgga

gggctgggcacacaggagctctgcattctggccccacttctttgcagaga

aggcctctactcctgagtccttgaaggacttgaggcccttgggagcctcc

Figure 3.14: genomic sequence of exon 9 showing the position of deleted nucleotide in green, the positon of added nucleotide in blue. ( www.ucsc.edu/ 20th August 2010,16.31)

The region of the query where the addition and deletion occurs was translated to protein sequence using expasy in order to know if there are changes to the protein sequence.

GTGA-CATCGGGCTGCCCACCAAGCAGCCCATCCCTGACTGTGAGATCAAGAATCGCCCG

AGCCTTCTGGTGGAGAAGATCAACCTGTTTGCCATGTTTGGAACTGGCATCGCCATGAGC

ACCTGGGTCTGGACCAAGGCCACGCTGCTCATCTGGAGGCGTACCTGGTGCAGGTGGGCA

TGGCAGCCAGCCCCTCCTGCCCTGCCCGCCTCACCCTCAGCCTTGGGACCCCATCTTTAG

GTTTTGTCGGGTCCTGCCTCTAGCACAGCCTTGGTCAGTGGTTCACCGCTGCCCCCTGGT

GGCACCTTCTGTCCTTGGGTGGCCTGATGCCTGGGCCTGGGCCTGGGCCAAGGGCAGAGC

CAGCTGCCATCACCTTTTAAGAAGCGGAGTGATTGGAGGGCTGGGCACACAGGAGCTCTG

CATTCTGGCCCCACTTCTT

Figure 3.15: The region of query that corresponds to exon 9 of the gene. The alteration to the sequence has been underlined. ( http://blast.ncbi.nlm.nih.gov/Blast.cgi 25th August 2010, 16.00).

5'3' Frame 3

D I G L P T K Q P I P D C E I K N R P S L L V E K I N L F A Met F G T G I A Met S T W V W T K A T L L I W R R T W C R W A W Q P A P P A L P A S P S A L G P H L Stop V L S G P A S S T A L V S G S P L P P G G T F C P W V A Stop C L G L G L G Q G Q S Q L P S P F K K R S D W R A G H T G A L

Figure 3.16: Translation of exon 9 to amino acid sequence (http://expasy.org/cgi-bin/dna_aa 26th August 2010,17.05)

The amino acid sequence was then blasted, giving the result below.The result indicate there is 100% match to human SMO gene meaning there is no single nucleotide polymorphism and the alteration i.e. the addition and deletion did not change the protein sequence.

sp

Q99835

SMO_HUMAN

Smoothened homolog precursor (SMO) (Protein Gx) [SMO] [Homo sapiens

(Human)]

787 AA

Score = 147 bits (333), Expect = 2e-34

Identities = 56/56 (100%), Positives = 56/56 (100%)

Query: 2 IGLPTKQPIPDCEIKNRPSLLVEKINLFAMFGTGIAMSTWVWTKATLLIWRRTWCR 57

IGLPTKQPIPDCEIKNRPSLLVEKINLFAMFGTGIAMSTWVWTKATLLIWRRTWCR

Sbjct: 496 IGLPTKQPIPDCEIKNRPSLLVEKINLFAMFGTGIAMSTWVWTKATLLIWRRTWCR 551

Figure 3.17: Amino acid sequence showing 100% identity with the human SMO gene in the data base.(http://www.expasy.ch/cgi-bin/blast.pl#A2 25th August 2010,17.34)

CHAPTER 4: CONCLUSION

This project was aim at understanding the role of Smo in normal cells and cancer, which had been achieved. The selection of a primer for the amplification of a particular region of the gene which had been implicated in some cancer disease, in this case exon 9 had also been achieved. The single nucleotide polymorphism observed in previous work changing codon 535 from Trp to leu (Xie et al, 1998) was not observed in this experiment. Further work might be needed to be done to confirm this mutation.

The deletion observed in exon 9 at base pair 5 might be due to error in the sequencing process but if it was real, it may be lead to a non functioning protein which may cause a disease.

The addiction that occurs at base pair 382 is located in the intron region, so does not have a significant effect.

CHAPTER 5 : FURTHER WORK

The sequence should be repeated to confirm what causes the deletion and addition in the sequence.

I recommend further works to be carried on exon 10 in which mutation has also been reported (Xie et al,1998)

Finally, i suggest a larger product size be use in order to have a detail view of possibility of single nucleotide polymorphism that may occur at larger base pair number.

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