LONG PCR AMPLIFICATION OF THE FVIII GENE INTRON 22 GENE INVERSION

Introduction

Long range PCR allows the amplification of PCR products, which are much larger than those achieved with conventional Taq polymerases. Up to 27 kb fragments are possible from good quality genomic DNA, although 10 - 20 kb fragments are routinely achievable, given the appropriate conditions. The method relies on a mixture of thermostable DNA polymerases, usually Taq DNA polymerase for high processivity (i.e. 5’-3’ polymerase activity) and another DNA polymerase with 3’-5’ proofreading abilities (usually Pwo). This combination of features allows longer primer extension than can be achieved with Taq alone.

This method for detection of the FVIII gene intron 22 inversion (Liu et al, 1998) removes the requirement for Southern Blotting. Results can be obtained within 24 hours. Modifications from standard long range PCR protocols include the addition of DMSO and incorporation of deaza GTP to enable read through of a high GC content region upstream of the FVIII gene. The method relies on overlapping PCR to generate a constant band, which appears in all template DNA’s. This band acts as a control to show that the reaction has worked efficiently. The largest amplification product seen using this method is 12 kb, well within the range of the enzyme mix utilised.

References:

Single tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in hemophilia A. Liu et al (letter) Blood 92, 1458-9, 1998.

Note that this reference contains a mistake in the primer B sequence (correct sequence given later in this protocol). For the correct primer sequences and some other useful tips if you suffer from over-amplification of some of the bands refer to:

Subcycling-PCR for multiplex long distance amplification of regions with high and low GC content: application to the inversion hotspot in the FVIII gene. Liu and Sommer Biotechniques 25, 1022-8, 1998.

Method Validation:

Several samples previously typed by Southern Blotting have been retested by the PCR method, giving concordant results. Six samples have been tested as part of a blinded NEQAS pilot scheme, giving the expected results.

This protocol was submitted by Steve Keeney, Haematology Department, Manchester Royal Infirmary. Any further enquiries should be addressed to:

skeeney@labmed.cmht.nwest.nhs.uk

REAGENTS

Oligonucleotide primers

1. INT22 P 5’-GCCCTGCCTGTCCATTACACTGATGACATTATGCTGAC-3’ (38 mer)

Make a 4 µM stock

2. INT22 Q 5'-GGCCCTACAACCATTCTGCCTTTCACTTTCAGTGCAATA-3' (39 mer)

Make a 4 µM stock

3. INT22 A 5’-CACAAGGGGGAAGAGTGTGAGGGTGTGGGATAAGAA-3’

(36 mer)

Make a 2 µM stock

4. INT22 B 5’-CCCCAAACTATAACCAGCACCTTGAACTTCCCCTCTCATA-3’

(40 mer)

Make a 2 µM stock

note: This primer B sequence differs from that published in the original letter which is incorrect.

Store stocks at -70^oC. Working stocks can be made by combining equal volumes of individual primer. If desired, these working stocks can be stored at -20^oC.

Repeated freeze-thawing of primers will reduce the efficiency of this protocol. Make small working stock aliquots of primer and dispose of after 2-3 freeze-thaw cycles.

dNTPs

Prepare individual stocks at a concentration of 10 mM in sterile water.

Ensure dNTPs are fresh – the freeze-thawing rule applies to these too.

deaza GTP

This is supplied by Boehringer Mannheim at a concentration of 10 mM.

Sub-aliquot this to minimise freeze-thawing.

Taq Polymerase-

Use the Expand “kit” from Boehringer Mannheim (Catalogue number 1681842). This comes with the appropriate buffer and Taq polymerase mix for optimal long template extension.

The mix includes a proof-reading Taq that will degrade single-stranded DNA, including primers. To prevent degradation, the Taq mix (master mix 1 - see below) needs to be kept separate from the primer containing mix (master mix 2) until immediately before thermal cycling.

PCR Buffer 2 (x10)

This is supplied with the Expand kit and is used at a final concentration of 1x in the reaction mix

An alternative to the Boehringer kit is the DyNAZyme EXT kit manufactured by Finnzymes. This kit is cheaper but you may encounter problems when using the supplied buffer. The DyNAZyme polymerase mix with Boehringer buffer 2 seems to work very well!

Sample Loading Buffer (5x)

4.8 ml glycerol

0.025g bromophenol blue (0.25%)

0.2 ml 20% SDS

5 ml 10x TBE

10x TBE

108g Tris

55g boric acid

40 ml 0.5M EDTA, _pH 8.0

to 1l with distilled water.

1kb BASE PAIR LADDERS (Gibco BRL)

AGAROSE

Use a molecular biology grade agarose, one with good gelling strength at low percentages.

DMSO

Use cell culture grade 100% DMSO

Sterile Water

METHOD FOR INTRON 22 LONG RANGE PCR

Samples

1. Extract DNA, determine concentration and use 0.1 to 0.25 µg per reaction. Too much DNA abolishes amplification. It may be advisable to carry out a DNA titration of a specific sample to ensure efficient amplification. If DNA is relatively concentrated then make a 1 in 10 dilution and use between two and five microlitres.

The quality of the DNA is paramount! Use an extraction method that does not shear the DNA and ensure that all traces of phenol are expelled if using phenol extraction. DNA prepared by ammonium acetate salting out methods works very well.

2. Always include a "no DNA" control in PCR

3. Always include a control heterozygous female carrier of the inversion.

Reaction Mix

Each reaction (final volume 25 µl) will require (In reality these will be made as a multiple master mix):

MASTER MIX 1 per reaction: MASTER MIX 2 per reaction:

2.5 µl 10x buffer 2 1.25 µl 10 mM dATP

0.94 µl Taq mix 1.25 µl 10 mM dTTP

5.69 µl water 1.25 µl 10 mM dCTP

0.625 µl 10 mM dGTP

0.625 µl 10 mM deaza GTP

5 µl P/Q primer mix (each at 4 µM)

3 µl A/B primer mix (each at 2 µM)

1.875 µl 100% DMSO

Total amount (set pipette value)

9.13 µl (9.2 µl) 14.875 µl (14.9 µl)

Allow each reagent to thaw fully, mix and spin down before pipetting.

Ignore the spurious accuracy of these figures! - when multiplied in a master mix they will be accurate.

For the above scheme make a master mix of all ingredients (excluding each DNA sample) X the number of samples to be amplified, including controls. This minimises pipetting losses of reagents and should decrease any tube to tube variability between reactions. The above volumes assume the addition of approximately 1 µl of template DNA to each reaction. If different amounts of template are used then adjust the volume of water in master mix 1 accordingly.

PCR

1. Aliquot master mix 2 / each individual DNA template first. Keep on ice.

2. Immediately prior to amplification step add appropriate volume of mix 1. Spin briefly and proceed to PCR step immediately.

3. Carry out PCR as follows:

TECHNE PROGENE

Cycling conditions:

Initial denaturing 94 C, 2 min no. of cycles

94 C, 10s - 65 C, 30s - 68 C, 12 min x10

94 C, 10s - 65 C, 30s - 68 C, 12 min + 20s per cycle x20

Final extension 72^oC, 5 min - Refrigerate

Cycling parameters/PCR machine used can affect the success of this protocol. See the additional notes at the end of this document for guidance.

4. Store samples at 4^oC prior to digestion.

AGAROSE GEL ELECTROPHORESIS

1. Prepare a 0.6% agarose gel:

0.48g agarose

80 ml 0.6 x TBE

0.5 µg/ml Ethidium bromide

Mix, melt thoroughly in microwave, cool to 60^oC, pour and allow to set.

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.