Protocol:CDNA sequencing

cDNA sequencing 

The method described below for the characterization of rare and unusual haemoglobin variants by cDNA sequencing is an unpublished method based on a modification of a technique previously reported by Liu et al (54). The technique described here allows the sequencing of transcripts of abnormal Hb genes for the identification novel variants in a five step process. Firstly, RNA is extracted from reticulocytes, then globin cDNA is synthesised by RT-PCR. Thirdly, the cDNAs for the -gene, the 1-gene and the 2-gene are amplified individually using specific primers. The amplified products are quantified by agarose electrophoresis and cleaned up to make ready for sequencing. Finally direct sequencing of the cDNA products is performed by cycle-sequencing. This approach permits the whole of the coding region of each gene to be screened for point mutational changes in a single sequencing run.

Preparation of RNA 

Total RNA is extracted from reticulocytes using a method based on the single step method of Chomczynski et al (55). Whole blood is centrifuged to band the reticulocytes which are then removed and RNA extracted using a commercial kit for ease of preparation such as RNA STAT-50 TM LS from Biogenesis Ltd, (Poole, England), but any kit for mRNA preparation would suffice.

Tips for success: ensure that all plastics and solutions are RNAase free, use RNAase free filter tips, and change gloves regularly throughout the extraction process


 * 1) Spin down 10 ml of whole blood with 2x volume of reticulocyte saline and collect approx 500 ml of reticulocytes from just below the buffy coat into an autoclaved 1.5ml eppendorf tube. Take great care not to include the buffy coat.
 * 2) Add 0.75 ml of RNA STAT-50ä (order from Biogenesis, catalogue # CS114, Fax # 01202 660020) vortex vigorously for at least 15 seconds [safe to store at –70 °C indefinitely]
 * 3) Add 200 ml of chloroform and vortex again for at least 15 seconds
 * 4) Cenrtifuge at full speed, (14,000 rpm) for 15 minutes at 4 °C
 * 5) Aspirate top aqueous layer into a new 1.5 ml eppendorf tube.
 * 6) Add 0.5 ml iso-propanol. Vortex well and allow to stand at room temperature for 10 minutes
 * 7) Centrifuge at 14,000 rpm for 10 minutes at 4 °C
 * 8) Tip off supernatant and discard.
 * 9) Add 1 ml 75% ethanol, vortex and spin at 14,000 rpm, 4 °C for 5 min
 * 10) Take off supernatant using a pipette, dry at 37 °C for 15 min
 * 11) Add 20 ml RNAase/DNAase free dH20 (Promega)
 * 12) Label and store –70 °C

Reverse transcription

The globin mRNA is transcribed into cDNA by reverse transcription. Use a commercial kit from Promega, the Reverse Transcription System, as per the manufacturer’s instructions. The kit from Promega ( Southampton UK, catalogue # A3500) allows for 100 reactions.


 * 1) Set up the reaction mixture (20 ml) as follows:
 * 1) Set up the reaction mixture (20 ml) as follows:


 * 1) 25 mM MgCl2 4 ml
 * 2) 10 mM dNTP’S 2 ml
 * 3) RT Buffer 10x 2 ml
 * 4) RNA inhibitor 0,5 ml
 * 5) AMV RT 0.8 ml
 * 6) Oligo dT 1 ml
 * 7) nuclease free dH2O 7 .7 ml
 * 8) template RNA 2 ml


 * 1) Place in heating block at 42 °C for 15 mins
 * 1) Place in heating block at 42 °C for 15 mins

-gene cDNA amplification


 * 1) Set up the reaction mixtures A and B as detailed below
 * 1) Set up the reaction mixtures A and B as detailed below

2. Add 35 ml mix A + 10 ml of reverse transcribed RNA template (cDNA)

3. Overlay with mineral oil.

4. Set up PCR machine for the following programme:

1x 94 0C for 3 min (hot start)

29x 94 0C for 1 min, 60 0C for 1 min,72 0C for 1 min

1x 72 0C for 3 min

5. After 2 mins of hot start, add 5 ml of mix B to each tube.

6. Cycle to end of programme

7. Run 5 ml of 50 ml product on a 2% agarose gel,; b globin cDNA product is 584 bp.

8. Save remaining 45 ml to be cleaned using Amicon Filters.

1-gene cDNA amplification

The same protocol is used as described for b-gene cDNA amplification, except that the primers  #390  5’a common (forward) and  #391  3’a1 specific (reverse) must be used.


 * 1) Hot start reaction for 2 min, then add 5 ml of mix B to each tube.
 * 2) Cycle conditions:

1x 94 0C for 3 min

29x 94 0C for 1 min, 600 for 1 min,72 0C for 1 min

1x 72 0C for 3 min


 * 1) Run 5 ml on 2% agarose gel, product size is 541 bp
 * 2) Save remaining 45 ml to be cleaned using Amicon Filters.

a2-gene cDNA amplification

Use the same protocol as for the b gene cDNA amplification, except use primers  #390  5’a common (forward) and  #392  3’a2 specific (reverse)


 * 1) Hot start reaction for 2 min, then add 5 ml of mix B to each tube.
 * 2) Cycle conditions:

1x 94 0C for 3 min

29x 94 0C for 1 min, 54 0C for 1 min,72 0C for 1 min

1x 72 0C for 3 min


 * 1) Run 5 ml on 2% agarose gel; product size is 554 bp
 * 2) Save remaining 45 ml to be cleaned using Amicon Filters.

Primer sequences


 * 1) b gene cDNA forward
 * 1) b gene cDNA forward

 #331  5’- ACA TTT GCT TCT GAC ACA ACT GTG - 3’


 * 1) b gene cDNA reverse
 * 1) b gene cDNA reverse

 #334  5’- GCC CTT CAT AAT ATC CCC CAG TTT - 3’


 * 1) 5’a cDNA common forward
 * 1) 5’a cDNA common forward

 #390  5’- CTG GTC CCC ACA GAC TCA GA - 3’


 * 1) 3’a1 cDNA specific reverse
 * 1) 3’a1 cDNA specific reverse

 #391  5’- ACG GGG GTA CGG GTG CAG GAA G - 3’


 * 1) 3’ a2 cDNA specfic reverse
 * 1) 3’ a2 cDNA specfic reverse

 #392  5’- TTT ATT CAA AGA CCA GGA AG - 3’


 * 1) 3’ a1 cDNA reverse sequencing
 * 1) 3’ a1 cDNA reverse sequencing

 #393  5’- AGC CCC AGG GGG CAA GAA GCA T - 3’


 * 1) 3’a2 cDNA reverse sequencing
 * 1) 3’a2 cDNA reverse sequencing

 #394  5’- GCC GGT GCA AGG AGG GGA GGA G - 3’

Cleaning of PCR products
The amplified product should be cleaned up before cycle sequencing using, for example, AMICON Microcon filter devices.


 * 1) Label tubes and filters.
 * 2) Place filter blue side up into tube, push on well.
 * 3) Load 45 ml PCR product, no oil, onto the filter, add 50 ml dH2O spin at 5000 g for 3-4 min.
 * 4) Add 100 ml of dH2O and spin again at 500 g for 3-4 min.
 * 5) Add a further 100 ml of dH20 and spin again at 5000 g for 3-4 min.
 * 6) Label up new tubes, remove the filter device from the original tube and turn so that the white side is face up and push it well onto the appropriate newly labelled tube.
 * 7) Add 15 ml of dH2O onto now the white side of the filter and spin at 5000 g for 3-4 min.
 * 8) Add a further 10 ml of dH2O and spin again at 5000g for 3-4 min.
 * 9) Run out 2 ml of this ‘cleaned’ product on a 2% gel against 4 ml of (50 ng\ml) i.e 200 ng of lHind III and fX174 DNA markers to quantify how much to use in the cycle sequencing reaction. (Top band of lHind III = ~100ng, middle band of fX174 = ~22 ng)

Cycle sequencing
The PCR products can be directly sequenced on a Genetic Analyser using a dye terminator reaction kit according to the manufacturer’s instructions with a minor modification. 2 ml of 5 M betaine, a powerful denaturant, is substituted into the cycle sequencing reaction instead of water for the 1 and 2-gene sequence analysis.


 * 1) For each sample separate reactions for forward and reverse primers are required:

For b gene sequencing use  #331  (f) and  #334  (r)

For a1 gene sequencing use  #390  (f) and  #393  (r)

For a2 gene sequencing use  #390  (f) and  #394  (r)


 * 1) Set up the reaction mix (per sample) as in the table below:

2. Mix, spin down, and overlay with 10 ml of mineral oil.

3. Cycle sequence with the following programme:

[95 0C for 30 sec, 50 0C for 15 sec, 60 0C for 4 min] X25, pause at 10°C

4. Transfer each reaction mix, minus the oil to a fresh, labelled 0.5ml eppendorf.

5. To each tube add 35 ml dH2O, 5 ml 3 M sodium acetate and 150 ml of 95% ethanol

6. Mix well and put on ice for 10 min (no longer)

7. Spin at full speed for 25 min.

8. Tip off the supernatant, add 50 ml of 65% ethanol to each tube, vortex hard (20 seconds) and spin at full speed for 5 min.

9. Carefully take off the supernatant using a pipette and leave to dry (to remove the ethanol) in a hot block at 55 °C for 1hour.

The cycle sequencing reactions are now ready for running on a DNA sequencing machine. If they are not to be used straight away they can be stored in the freezer until required. If they are stored in the freezer, remember to put them in the hot block again at 55 °C for 20 min before proceeding further, this is to remove any condensation that may form on thawing, which may affect the reaction.

References


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