From: msalminen@nphi.fi
Newsgroups: bionet.molbio.methds-reagnts
Subject: Best direct sequencing protocol!?
Date: 6 Oct 92 16:55:32 GMT
Organization: National Public Health Institute, Finland

Hello netters!

I'd like to add my 5p to the discussion about the best way of sequencing PCR
products. Im sending you my protocol for solid phase sequencing of biotinylated
DNA. Ie you do your pCR with a biotinylated oligonucleotide, bind it to avidin
coated beads, remove the opposite strand by NaOH treatment and sequence the
ssDNA dirctly. It's rapid, simple and very reliable (If your pcr products are
clean; use Hot Start). I'm using it for HIV-1 direct sequencing and it works
like a dream. I use both nested, and the PCR primers for sequencing. Youll have
to test whats best. The manganese equalizes the T7 pols affinity for the
nucleotides, so you have very equal signal in all lanes.

However, it also increases the affinity of the enzyme for ddNTPs so you must
change the dd/dNTP ratios as below to be able to read >= 350 bp.

You will need 1 pmol of template per sample so youll have to do a calculation
of your PCR product but usually about 200-500 ng is enough if the fragment
lenght does not exceed 500 bp. The beads only bind about 1.6 pmol biotin per 10
ul so use 1pmol per pcr (5o ul reaction)

The sequencing protocol works well for all kinds of ss templates, ie M13 is
great (>450 bp)


If you have any questions, please feel free to ask, i hope you enjoy the
protocols! ;-)

		Regards, Mika
		msalminen@nphi.fi



Disclaimer: I have no connection whatsoever with the commercial companies
mentioned below.


                        Affinity capture of Bio-DNA

              (c) 1991, 1992 Mika Salminen FINNPHI, HIV-Unit.
                             msalminen@finnphi

ref: Salminen, Mika. Rapid and simple solid phase direct
sequencing of in vivo HIV-1 quasispecies. (1992) AIDS Research
and Human Retroviruses, 8;1747-1756. 



        1.       Dilute x ul BIO-PCR 100 ul:s in TENT 
        2.       Add 10 ul 5% (w/v) avidin-polystyrene beads
		 (IDEXX, cat nr31-040-1, fax 207-856-0346).
                 Inkubate RT, 45-60 min. --> cfg. 3 min, 6.000
                 rpm (eppendorf), remove sup with gentle
                 suction.
        4.       Denature 50 ul 50 mM NaOH 5 min RT --> cfg. 3
                 min, 6.000 rpm, sup away (neutralize with HCl
                 and ppt if you want to seq. opp. strand)
        5.       Wash twice TENT --> cfg. 3 min, 6.000 rpm,
                 discard sup.

Needed buffers:

        Den.     50 mM NaOH  -> 100 ml Milli-Q H20 + 500 ul   
                 5 M NaOH, Sterilize. Keep at + 4 C.
        TENT     0.01 % Tween 20, 1 mM EDTA, 50 mM Nacl, 40 mM
                 Tris HCl pH 7.5.
        ->       200 ul 0.5 M EDTA (pH 8) + 4 ml 1 M Tris-HCl
                 pH 7.5 + 0.2922 g NaCl, Sterilize. Add 
                 sterile 10 ul Tween-20. Keep at + 4 C.

                          SEQUENASE 2.0 SEQUENCING
                         ON MIKROTITERPLATES, with Mn2+

              (c) 1991, 1992 Mika Salminen FINNPHI, HIV-Unit.
                             msalminen@finnphi

ref: Salminen, Mika. Rapid and simple solid phase direct
sequencing of in vivo HIV-1 quasispecies. (1992) AIDS Research
and Human Retroviruses, 8;1747-1756. 

REAGENT-POOLS (8 samples):

        B/P = Buffer/Primer mix

        Kit reagent                9 * pool (1 *)

        5 * Seq. reaction buff.    18        (2)  
        1 uM Primer                45        (5)
        Ster H2O                   27        (3)
                                   90       (10)

        L/M = Labelling mix

                                    9 *      

        1:5 dGTP Lab. Mix            18   (2)
        DTT 0.1 M                     9   (1)
        100 mM Mn2+ buffer            9   (1)
        Ster H2O                    4.5 (0.5)
                                   40.5 (4.5)

Make pools while BIO-DNA binds to IDEXX-beads. Pipet 4.5 ul L/M
mix per well on the mikrotiterplat according to the graph below.
Handle on ice, plate also.


        L = dATP-35-S Label

Amersham freeze-dried, one lane = 8 samples. Keep on ice on
separate plate.


        A, C, G, T - Termination/extension mix

Own termination mixes, (with 7cdGTP) marked A-T. Pipet 3 ul
each in own wells according to the graph below (on ice).


        Sequenase 2.0 entsyme

Dilute 1:3 when hybridization step ower !!!Stock-entsyme must
be kept in -20 C freezerblock all the time!!!, dilution on ice
all the time.

        User dilution               9 *     

        Enz. Dil. Buffer           13 ()
        Sequenase 2.0 stock         6 ()
                                   19 (2)

Diluted enzyme is added (2 ul per sample) as hybridized
Template/primer complex has been mixed to labelling mix. 


                           REAGENT ORDER ON PLATE




                                                                
                     1  2  3  4  5  6  7  8  9  10 11 12        
                          L/M    A  C  G  T            S        
                                                                
                                                                
                 A                                              
                                                                
                 B                                              
                                                                
                 C                                              
                                                                
                 D                                              
                                                                
                 E                                              
                                                                
                 F                                              
                                                                
                 G                                              
                                                                
                 H                                              
                                                                
                                                                

Ready on plate:

                 Label, one eight well row, on separate plate
                 (holder)

                 L/M = 4.5 ul per well

                 A = 3 ul per well

                 C =      -"-

                 G =      -"-

                 T =      -"-

                 S = 20 ul STOP buffer (Sequenase)


                       DOING THE SEQUENCING REACTIONS

        HYBRIDIZATION:

        Resuspend bound ssBIO-DNA (Beads) 10 ul B/P mixi . -->
        + 37 C 10 min, RT 10 min.

        LABELLING:

        Move BIO-DNA:t through L-wells to LM-wells. Pipet
        setting 14,5 ul, pipet back and forth in wells 3-4 tms.
        to mix thoroughlly. When all BIO-DNA:s moved, add 2 ul
        entsyme fast (diluted) per well. Pipet back and forth
        with 8-channel pipet (setting 10 ul) a couple of times,
        beware of bubbles. Inkubate RT 4 min.

        EXTENSION/TERMINATION

        Move 4 ul from each LM-well to each A,C,G, ja T-well. 
        Change tips between wells. Mix by pipettin 3-4 tms. -->
        +37 5 min (-15 min)

        STOPPING OF REACTIONS:

        Add 4 ul loading buffer from the S well to the
        termination wells. Change tips between wells. Mix by
        pipetting. Keep covered with tape at -20 C until you
        run gel. Denature +75 C, 3 min --> ice. Load 2-5 ul
        depending on the gel.

        THINGS TO REMEMBER:

        - Use only high quality precicion tips and pipettors.
        - Change tent buffer once a month 



Custom Mn2+ termination and labelling mixes.

Labelling mix

1,5 uM each dNTP (except labelled nucleotide, A in this case)

Pre-dilution:

100 mM Pharmacia Ultrapure dCTP         1 ul
                           dGTP         1 ul
                           dTTP         1 ul
                           sH2O       497 ul
                                      500 ul = 200 uM dATP Lab. Mix
Final Dilution:

200 uM dATP lab. Mix        10 ul
               sH2O       1320 ul
                          1330 ul = 1,5 uM dATP Lab. Mix

Termination mixes (with 7cdGTP)

1 mM dNTP + 5 uM resp ddNTP
                        
Pre-dilution: Pharmacia ddNTP = 5 mM => dilute 1:100 = 2 ul + 98
ul sH2O = 50 uM

Final dilutions:

                             A    C    G    T  
                                                            
Pharmacia 5 mM 7cdGTP       40   40   40   40   (ul)
        100 mM   dATP        2    2    2    2  
        100 mM   dCTP        2    2    2    2  
        100 mM   dTTP        2    2    2    2  
                                               
        50 uM   ddATP       20   -    -    -   
        50 uM   ddCTP       -    20   -    -   
        50 uM   ddGTP       -    -    20   -   
        50 uM   ddTTP       -    -    -    20  
                                               
                 sH2O      134  134  134  134  
                                                            
                           200  200  200  200