DNA Sequencing

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 Sequencing of genes was performed using the following methodology. The exons together with the intron-exon boundaries were sequenced and screened for any SNPs and/or other mutations that might be present. Sequencing was initially performed in the most severely affected family members and both related and unrelated normal controls. Individuals having lumbar and/or femoral t-scores >1.5 were selected as normal controls (with normal BMI). Any variants identified were then analysed in all affected family members. DNA sequences of genes were retrieved from the public database ENSEMBL (http://www.ensembl.org). Oligonucleotide primers were designed using the freely available software OligoExplorer v1.2 to yield PCR fragments of not more than 630 bp. The fragments were amplified by PCR in a 50 μl total reaction volume, containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 200 μM of each dNTP (Promega, Madison, WI, USA), 50 ρmol of each primer and 100 ng genomic DNA. The fragments were then checked by 1% agarose gel electrophoresis and photographed using a Polaroid camera.

 

Cleaning of PCR products

Following PCR, amplified products were cleaned from any nucleotides, primer dimers and primers that were not utilised during the reaction. Cleaning was performed using Microcon Y-100 (Millipore Corporation) centrifugal filter units that remove any fragments of less than 100 bp.  The following protocol was performed at room temperature, by first adding 300 μl of sterile distilled water to each empty filter device and centrifuged for 10 minutes at 300 g. Following centrifugation the PCR product together with 50 μl of sterile distilled water were added to the filter device. Centrifugation was carried out for 10 minutes at 500 g, followed by the addition of another 30 μl of sterile distilled water. After leaving the filter to stand for 3 minutes, it was inverted and placed in a new sterile 1.5ml microcentrifuge tube. Centrifugation was performed at 1,000 g for 3 minutes, after which the filter was discarded and the cleaned product was stored at -20°C until analysed.

 

 

Cycle Sequencing and electrophoresis

Both forward and reverse sequencing of the exon regions of the genes was performed using the BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Two microlitres of cleaned PCR product were added to the reaction mix containing 2μl of BigDye® mix, which contains the four fluorescently labelled dideoxynucleotides and polymerase, 2 μl sequencing buffer and 4 ρmoles of forward or reverse primer. The sterile water was added up to a total reaction volume of 10μl. The PCR reaction was carried out using a GeneAmp® PCR system 9700 (Applied Biosystems, Foster City, CA, USA) using the following thermal profile. An initial denaturation step was carried out at 96°C for 1 minute followed by 25 cycles at 96°C for 10sec, 50°C for 5sec and 60°C for 4 minutes. A pGEM -3Zf(+) sequencing control was performed with each run of sequencing. The samples were cooled rapidly to -20°C and kept at this temperature until analysed.

Following cycle sequencing, excess dye terminators not utilised in the reaction were removed prior to electrophoresis using the genClean Dye Terminator cleaning kit (Genetix Limited, Hampshire, UK).  This step was carried out to prevent excess dye terminators from obscuring the data. The genClean columns were first mixed and centrifuged at 1,000 g for 3 minutes to remove the storing buffer leaving only a gel matrix. Following centrifugation, 200μl of sterile distilled water were added to the columns followed by another centrifugation for 3 minutes at 1,000 g. The columns were transferred into clean 1.5ml centrifuge tubes and 15μl of sterile distilled water were added to the sample to be sequenced before transferring onto the centre of the gel in the cleaning columns. The columns were centrifuged at 1000 g for 4 minutes. The cleaned products to be sequenced were collected in the 1.5ml centrifuge tubes while the columns containing the gel matrix with unincorporated dye terminators were discarded.

Fifteen microlitres of cleaned products were transferred into a 96 well plate and denatured at 95°C for 3 minutes followed by rapid cooling on ice. The samples were then subjected to capillary electrophoresis using an ABI 3130 genetic analyzer (Applied Biosystems, Foster City, CA, USA) with a 36cm capillary and POP7 as polymer. Base calling was performed using the Sequencing Analysis Software v5.2 (Applied Biosystems, Foster City, CA, USA). The sequencing results obtained were compared with reference sequences obtained from public databases using ChromasPro Version 1.33 (http://www.technelysium.com.au). Using this software one can directly compare sequencing results with sequences on the NCBI database (http://www.ncbi.nih.gov/).

Forward and Reverse Sequencing Primers for TRAF6 Gene found in locus 11p12 (Transcript ENST00000313105)

 

Primer Sequence (5` - 3`)

Fragment Size (bp)

Annealing temp (°C)

TRAF6 – Prom1*

CAA ATT CCT GGC CTC AAG TGA

331

56

TGG TTC ATC GTC CAC CTC TTA G

TRAF6 – Prom2*

CCC TAA GAG GTG GAC GAT GAA

357

56

CTC CAA GCA GGA GAA AAC CC

TRAF6 – Prom3*

CCA GCT GGG GTT TTC TCC T

215

56

GCA GCC TGG CTT TCT TCC T

TRAF6 – Exon 1

ACC CGA GCA GGA AGA AAG C

552

50

CCC TGA CCT GTG AAA AGC G

TRAF6 – Exon 2

CTA TTC CCT TCA GCT CAG GAT AC

597

48

CAC ACA GCA GTC ACT TTC AGA C

TRAF6 – Exon 3

GAT CCA GCC AGT CTG AAA G

475

45

GTC AAG AGA GGA GGA GAT TC

TRAF6 – Exon 4

GTG AGC AGC ATA TTC CAT C

617

45

ACA CAG CAA AGT CCC TAT TC

TRAF6 – Exon 5

GAG TTG GCT AAT GCT AAT CC

300

45

AGT CGG AGT CAC ATC CTT ATC

TRAF6 – Exon 6

TAT AGC CTC GGC AGA TGA TG

578

51

GGG CTG GAT GTT TTG TTC TC

TRAF6 – Exon 7

GGG AGT CTT AAT CCA GTT TG

585

45

GCT AAG TAT GCC TTT GCT TC

TRAF6 – Exon 8a**

CAG AGT CGC CTC ATT GGA TC

586

50

GCG CAT GCA CAG TTT GTA CC

TRAF6 – Exon 8b**

GAC AAG ACC ATC AAA TCC G

630

45

TAC TTC GTG GCT GAA AAC C

TRAF6 – Exon 8c**

ATC AGT CTG AAG CAC CTG TAA G

600

50

ACT CTT GAG TCT GGA CTT TCT G

TRAF6 – Exon 8d**

GTA CTT TCT TGG GCT TTT GCT C

438

50

GGA AGA TGC TAC TTC GTA ACC TC

*Solution S (Solis BioDyne , Tartu, Estonia) was added to the PCR reaction

**Exon 8 was initially divided into 4 overlapping fragments. Due to the presence of a pseudogene on chromosome 10 with a similar sequence to part of fragments 8b and 8c, a single large fragment of 1.3kb was amplified by PCR using the forward primer of fragment 8b and reverse of 8d using an annealing temp of 45°C. This was done to prevent the pseudogene from being amplified together with exon 8 of the TRAF6 gene. Sequencing was performed using the above primers.

 

   
     
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