LONG PCR AMPLIFICATION OF THE FVIII GENE INTRON 22 GENE INVERSION
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Adjust according to your apparatus. Thin combs will improve band resolution.
2. Add 5µl of 5x loading buffer to each 25 µl PCR product and load 5-10µl on the gel (see below)
3. Load 1kb ladder (Gibco-BRL or equivalent)
4. Run the gel at 90 volts for 5 hours.
5. Visualise bands on gel using UV transilluminator and photograph
Interpretation of banding pattern:
An upper 12 kb band and a lower 10 kb band only means the inversion is not present.
A middle 11 kb band and a lower 10 kb band means the inversion is present in an affected male.
Bands at 10, 11 and 12 kb indicate a female carrier of the inversion.
Click
HERE for an image of the expected patterns.
Problems with visualisation of the bands may include overamplification, leading to smearing on the gel. If this is a problem load less. If selective over-amplification occurs, obscuring particular target bands, then refer to the Liu and Sommer 1998 reference for tweaks to the method.
METHOD NOTES
Key points when designing a long range PCR protocol:
· The template DNA must be of a good quality. Degraded or sheared DNA will not amplify. For human genomic DNA this equates to a band which runs on a gel with a size estimate of >50 kb. Freshly extracted DNA works very well. Archived samples may give fainter amplification.
· PCR primers need to be longer (24-34 bp) and have a higher, balanced, melting temperature (63-68 degrees is optimal) This allows higher reaction specificity.
· Cycling conditions are critical. Faster ramp rates on your PCR machine will improve the quality of amplification. Denaturation times need to be kept to a minimum (12 seconds or less is recommended) in order to avoid degradation of newly synthesised product. Thin-walled PCR tubes must be used to minimise thermal lag. 200 microlitre PCR tubes may improve the quality of amplification. Extension times are long and need to be extended for each additional cycle. In the case of the FVIII gene intron 22 long PCR the extension time for a 12 kb product begins at twelve minutes for the first cycle and reaches 19 minutes by the thirtieth cycle.
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