Polony Experimental Protocol Manual v1.1

 

Church Lab

Department of Genetics

Harvard Medical School

 

Mitra Lab

Department of Genetics

Washington University in St. Louis

 

Contributors

 

 

Last Modified 11-22-02 by Jay Shendure

 

 

 

Table of Contents

 

 

I. Introduction to Polonies

 

 

II. Reagents & Equipment

 

 

III. Experimental Protocols

 

A.     Subsidiary Protocols

 

B.    PCR Amplification

 

C.    Genotyping

 

D.    Haplotyping

 

E.     Exon Typing

 

F.     Multi-Base Sequencing

 

 

IV. Appendixes

 

A.     Primer Design

 

B.    Software

 

 

V. REFERENCES

 

 

 

I.                    Introduction to Polonies

 

The purpose of this manual is to concisely provide all of the necessary information to get the polony protocol up and running for individuals or laboratories new to the technology.  It also provides a working document which we can use as a reference for “what works” but simultaneously allow to evolve over time as protocols and software develop.  We are committed to open-source protocols and software, and will endeavor to use off-the-shelf equipment wherever possible.  Contributions of anecdotes and/or suggested modifications or clarifications are by all means welcomed.  We have tried to be very specific about which reagents and equipment were used by us in developing this protocol.  We strongly urge users to stick to this reagent & equipment list, and only make substitutions after having clearly established a working implementation of the protocol.

 

References 1-3 provide general and technical background on polony amplification, haplotyping, and sequencing.

 

 

II.                  Reagents & Equipment

 

All but the last sub-section describe reagents and equipment for PCR amplification and single-base-sequencing, which should necessarily be a stepping-stone to establishing a multi-base sequencing protocol.  The last sub-section lists reagents specific to the multi-base sequencing protocol.

 

Gel Components

 

·        40% IEF Acrylamide (Pharmacia; 17-1301-01)

·        BIS (2% Methylenebisacrylamide) (Pharmacia; 17-1306-01)

·        DATD (Aldrich; 15,686-8)

·        Acrylamide / Bis (19:1; 38%:2%) (Roche; 1-814-290)

·        APS (Sigma; A7460)

·        TEMED (Sigma; T-9281)

 

Slide Related

 

·        Teflon Coated Glass Slides (Erie Scientific; 285W)

·        Glass Cover Slips (18 mm x 30 mm; need to call Fisher Scientific and custom order; ~$300 for 5 cases)

·        FrameSeal Chambers (MJ Research; 125 uL; SLF-1201)

·        SecureSeal Chambers (Grace Biolabs; SA500; 10484915; 40x22x0.8 mm; Schleicher & Schuell Bioscience sells the same item for 1/3 the price of Grace Biolabs)

·        Plastic Coplin Jars

·        Glass Coplin Jars

·        Black Box Slide Racks

·        White Slide Holding Boxes

·        Glass Coplin Buckets

·        Metal Slide Racks

 

Reagents

 

·        Jumpstart Taq w/ 10x buffer (Sigma; D6558; with MgCl2 in 10x buffer)

·        30% BSA (Sigma; A7284)

·        Klenow (3-5’ exo-minus) polymerase (NEB; M0212M; SPECIAL ORDER HIGH CONENTRATION; 50,000 UNITS / uL)

·        SSB (USB; 70032Z)

·        Bind Silane (Pharmacia; 17-1330-01)

·        SYBR Green I (Molecular Probes; S-7563; 10000x)

·        10 mM unlabeled dNTP mix

 

Solutions

 

·        Hexane (Sigma; H9379)

·        1 M Tris-Acid (make with Tris-Hydrochloride; JT Baker; 4103-02)

·        1 M Tris-Base (make with Trizma Base; T6066)

·        Iodoacetamide (Sigma, I-1149)

·        2-mercaptoethanol (Sigma, M6250)

·        Acetic Acid (99.5% Glacial; Mallinckrodt; UN2789)

·        Formamide (Sigma; F-5786)

 

·        0.5x TBE

·        10% Tween

·        6x SSPE w. Triton X-100

·        20x SSC

·        Wash 1E (WB 2300)

·        Tritron X-100

 

Single Base Extension Nucleotides

 

·        1 mM Cy5-dATP

·        1 mM Cy5-dGTP

·        1 mM Cy5-dCTP

·        1 mM Cy5-dUTP

·        1 mM Cy3-dATP

·        1 mM Cy3-dGTP

·        1 mM Cy3-dCTP

·        1 mM Cy3-dUTP

 

·        Cy5-SS-dATP (special synthesis by Perkin-Elmer-NEN)

·        Cy5-SS-dGTP (special synthesis by Perkin-Elmer-NEN)

·        Cy5-SS-dCTP (special synthesis by Perkin-Elmer-NEN)

·        Cy5-SS-dUTP (special synthesis by Perkin-Elmer-NEN)

 

Equipment

 

·        PCR Hood

·        Scanner

·        Desiccation chamber

·        Slide Cyling PCR machine

·        Flat shaker at room temperature

·        Flat shaker in 70 degree C incubator

 

Miscellaneous Items

 

·        Tweezers (several pairs)

·        Razor Blades

·        15 mL polypropylene conicals

·        dH20 (autoclaved)

·        Sharpie Pens

·        0.22 micron filters

·        Syringes

·        Stir bar

           

 

III.                Experimental Protocols

 

This chapter has been divided into six sections.  The 1st section provides miscellaneous protocols and recipes for wash solutions and buffers.  The 2nd covers the protocol for single-molecule amplification in an acrylamide gel (e.g. polony PCR).  The 3nd through 6th sections cover protocols for various queries that can be performed on amplified DNA; respectively, genotyping, haplotyping, exon-typing, and sequencing.

 

A. Subsidiary Protocols

B. PCR Amplification

C. Genotyping

D. Haplotyping

E. Exon-Typing

F. Multi-Base Sequencing

 

 

A. Subsidiary Protocols

           

Stock Solutions

 

-         2M KCl

-         500 mM EDTA (pH 8.0)

-         1M MgCl

-         1M Tris (pH 7.3)

-         1M Tris (pH 7.5)

-         1M Tris (pH 8.0)

-         1M Tris (pH 8.8)

-         dH20

           

Special Buffers

 

-         WASH 1E (WB 2300)

-         1X Klenow Extension Buffer

-         1x Bleaching Buffer

-         1x Alkylation Buffer

-         1x Quenching Buffer

-         20x SSC

-         6x SSPE w. Triton X-100

           

Special Protocols

 

-         Bind-Silane Treatment of Slides

-         Repel-Silane Treatment of Slides

-         CYBR Green Staining of Acrylamide Gels

-         A-B-T MIX

 

 

 

WASH 1E (WB 2300)

 

Final Formulation:

 

10 mM Tris (pH 7.5)

50 mM KCl

2 mM EDTA (pH 8.0)

0.01% Triton X-100

 

Recipe for 1 liter:

 

(1) Mix the following

 

10 mL 1M Tris (pH 7.5)

25 mL of 2M KCl

4 mL of 500 mM EDTA (pH 8.0)

 

(2) Fill to 1 liter with dH20

 

(3) Add 100 uL of Triton X-100 (to 0.01%)

 

Mix by shaking or with stir-bar

 

Wash 1E can be stored at room temperature.

 

 

1X Klenow Extension Buffer

 

Final Formulation:

 

50mM Tris (pH 7.3)

5mM MgCl2

0.01% Triton X-100

 

Recipe for 1 Liter:

 

(1) Mix the following

 

50 mL of 1M Tris (pH 7.3)

5 mL of 1M MgCl2

 

(2) Fill to 1 liter with dH20

 

(3) Add 100 uL of Triton X-100 (to 0.01%)

 

Mix by shaking or with stir-bar

 

1X Klenow Extension Buffer can be stored at room temperature.

 

 

1x Bleaching Buffer

 

Final Formulation:

 

100mM Tris (pH 8.8)

 50mM KCL

 

Recipe for 1 Liter:

 

(1) Mix the following

 

100 mL of 1M Tris (pH 8.8)

25 mL of 2M KCl

 

(2) Fill to 1 liter with dH20

 

Mix by shaking or with stir-bar

 

Bleaching buffer can be stored at room temperature.

 

 

1x Alkylation Buffer

 

Final Formulation:

 

300mM Tris (pH 8.0)

50mM KCL   

 

Recipe for 1 Liter:

 

(1) Mix the following

 

300 mL of       1M Tris (pH 8.0)

 25 mL of        2M KCl

 

(2) Fill to 1 liter with dH20

 

Mix by shaking or with stir-bar

 

Alkylation buffer can be stored at room temperature.

 

 

1x Quenching Buffer

 

Final Formulation:

 

10mM Tris (pH 7.5)

50mM KCL

20mM EDTA

 

Recipe for 1 Liter:

 

(1) Mix the following

 

       10 mL            1M Tris (pH 7.5)

       25 mL            2M KCl

       40 mL            500mM EDTA (pH 8.0)

 

(2) Fill to 1 liter with dH20

 

Mix by shaking or with stir-bar

 

Alkylation buffer can be stored at room temperature.

 

 

20X SSC

 

See MANIATAS manual (B.13 of 2nd Edition) for 20X SSC recipe

 

 

6X SSPE w. Triton X-100

 

See MANIATAS manual (B.13 of 2nd Edition) for 20X SSPE recipe

 

Adjust to 6X with dH20.

 

Add Triton X-100 to 0.01%

 

 

Bind-Silane Treatment of Slides

 

All slides must be pre-treated with Bind-Silane prior to use.  We typically treat several hundred slides at a time, and store the slides in a dessicator.  Treating slides requires a few hours of time but only a small amount of hands-on work. 

 

  1. Load slides (facing the same direction) into metal slide racks

 

  1. Sterilize the following items (15 minutes under UV light in PCR hood):

 

    1. two glass Coplin buckets & lids
    2. metal slide racks with slides loaded
    3. 1 L dH20
    4. stir bar

 

  1. Add 220 uL Acetic Acid to 1 L dH20 (pH -> 3.5; no need to measure)

 

  1. Add 4 mL Bind-Silane

 

  1. Mix for 15 minutes (using stir-bar)

 

  1. Place slide racks in glass Coplin buckets

 

  1. Add stirred mixture to Coplin buckets (~500 mL per bucket; enough to cover the slides)

 

  1. Cover buckets and incubate with slow shaking for 1 hour

 

  1. Rinse each slides/bucket 3x with dH20

 

  1. Rinse each slides/bucket 1x with ethanol

 

  1. Remove slide-racks from buckets and allow slides to dry (in racks) under PCR hood

 

  1. Transfer slides to white boxes (facing the same direction)

 

  1. Label boxes with initials and date of treatment

 

  1. Place into vacuum dessicator for long-term storage

 

Notes:

 

Try not to touch surfaces of slides while transferring to and from racks

 

Place paper towels down wherever you are placing buckets or racks (e.g. shaker) to keep things clean.

 

 

 

Repel-Silane Treatment of Slides

 

1.            Use a chem-wipe to apply Repel-Silane to individual slide surfaces

 

2.            Let stand for 10 minutes

 

3.            Submerge in EtOH and put on shaker for 3 minutes

 

4.            Dump EtOH and suberge in dH20

 

5.            Put on shaker for 3 minutes

 

6.            Remove slides & let dry under UV

 

 

 

CYBR Green Staining of Acrylamide Gels

 

CYBR Green staining is not something that we routinely do.  It stains dsDNA, and can be used to check whether polony amplification worked without going through a single or multi-base sequencing protocol.  However, the signal-to-noise ratio is so poor, and the images so messy, that we strongly prefer performing a single-base-extension even for simply checking how well a polony amplification worked.  Nevertheless, this protocol can be useful for trouble-shooting.

 

  1. Mix 8 uL of concentrated CYBR Green (10000x) into 50 mL 0.5X TBE.

 

  1. Incubate slides with mix in plastic Coplin jar on shaker for 20 minutes.

 

  1. Wash once with 0.5X TBE (20 minutes on shaker in plastic Coplin jar).

 

 

A-B-T MIX

 

Lately we have been trying out pre-mixing the acrylamide, bis and DATD.  This is not what is done in the protocols below, but I’m just putting to protocol for the mix in here in case it eventually becomes generally adopted.  It makes life a lot easier, as solution making steps and argon degassing steps are eliminated.

 

To make 50 mL:

 

1 gram DATD

5 mL 40% Acrylamide:Bis (19:1) [Roche]

45 mL 40% IEF Acrylamide

 

Wrap container in tin-foil and store at 4 degrees C.  For use, simply run a small amount through a 0.22 micron filter immediately prior to making gel mix.  Use as ~15% of total polymerization mix for a 6% gel.

 

 

B. PCR Amplification

 

This section outlines the protocol for single-molecule amplification within the acrylamide gel, an obvious prerequisite to sequencing.  The following sub-sections are provided:

 

-         CAST ACRYLAMIDE GELS

-         REAGENT DIFFUSE-IN

-         PCR AMPLIFICATION

-         SLIDE CLEAN UP

 

Notes

 

1.                     Slides must be Bind-Silane treated prior to use.  See Subsidiary Protocols.

 

2.                     It is strongly recommended that both a lab area and a freezer area be designated as  “PRE-PCR”.  Post-PCR slides and post-reaction PCR tubes should always be kept away from the PRE-PCR designated areas.  We are performing single-molecule PCR, often with ‘common’ primers, so a small amount of contamination can go a long ways towards uninterpreteble or misleading results.  We have had little to no problems with cross-contamination since designating PRE-PCR areas.

 

3.                     Always include one slide as a no-template negative control.  This is a means of quickly determining when a cross-contamination has become a problem.

 

4.                     The below protocols assume that 9 slides are being processed, and the volumes, etc. are specified accordingly.  This is a particularly convenient number of slides to process because the plastic Coplin jars can fit nine slides.  We have successfully processed up to 32 slides in a single run, but suggest starting with 9 and scaling up once everything is working.

 

5.                     Try not to come into contact with oval-region of glass slides.  Best to keep it clean as possible both before and after Bind Silane treatment.

 

6.                     An argon chamber can be as simple as a Ziplock bag with a tube fitting such that it can be filled with argon gas.

 

7.                     It is quite possible to include all PCR reagents in the gel from the get-go, such that a diffuse-in is not necessary.  However, we have found that PCR amplifications are considerably more efficient when as many PCR-related reagents as possible are diffused in after the gel has polymerized (probably because the polymerization process can damage DNA and/or enzymes).  In the protocol described below, template is included in the gel during polymerization.  It is also quite possible to diffuse-in template (and this may lead to even better amplificaton results).  However, the distribution of polonies across the space of the gel can be highly non-uniform.  Consequently, we still often elect to mix the template into the polymerization mix.

 

8.                     In the convention that we adopt for this protocol, the forward primer (FP) and the sequencing primer (SP) are in the same orientation with respect to the desired PCR product, while the reverse primer (RP-AC) obviously is in the opposite orientation.  Unless otherwise specified, it is always the reverse primer that is bearing the acrydite-modification that will immobilize PCR product to the acrylamide gel (thus the “AC” after “RP”).

 

 

CAST ACRYLAMIDE GELS

 

7.      Turn on UV lamp in PCR hood for 15 minutes prior to use.

 

8.      Take 9 Bind Silane treated slides out of dessicator and place face up under hood.

 

9.      Label slides with identity and date using a SHARPIE pen (other inks will wash off in hexane)

 

10. Almost completely cover slides with coverslips (Fisherbrand, 18mm x 30mm, #1, untreated).  Keep a small area of oval exposed for subsequent gel loading.

 

11. Prepare fresh 2% DATD in a 15 mL conical.

 

40 mg DATD → 2 mL dH20

 

6. Argon degass 2 mL of each of these reagents:

 

-         dH20

-         2% DATD

-         40% IEF acrylamide monomer

-         2% BIS

 

This is essentially accomplished by lightly bubbling argon gas into each solution (in a 10 mL conical) for several minutes.  An alternative is to filter a small amount of each of these four reagents through a 0.22 micron filter.  The goal is to remove gas bubbles in each solution that may subsequently inhibit gel polymerization.

 

A simpler alternative that we are beginning to explore is to mix before hand the above four reagents at a specified set of ratios, store a stock mixture at 4 degrees C, and use argon-gas or filter on a small amount of this mix at this step.

 

7. Prepare fresh 5% APS.  Notably, APS should be stored in some sort of room-temperature dessicator.

 

0.5 g APS →10 mL dH20

 

8. Prepare fresh 5% TEMED

 

2 uL TEMED → 38 uL dH20.

 

9. Make 250 uL Acrylamide Gel Master Mix.  Note that TEMED and APS (and template, assuming that it is unique to each slide), are not added to the master mix.

 

 

Note: RP-AC = “Reverse-Primer, Acrydite Modified”

 

10. Aliquot 23 uL of Master Mix into each of 9 tubes.

 

11. Gel Loading: Process slides individually to ensure that gel is loaded before onset of polymerization.  Add unique template (1 uL), TEMED (0.5 uL), APS (0.5 uL) to each tube.  Load 18 uL into exposed area of oval.  Liquid should distribute under coverslip such that only a small amount (~1 uL) cannot fit.  Slide coverslip over such that oval is completely covered.

 

12. Place tray of slides into argon-filled chamber.  Allow gels to polymerize over 30 minutes.

 

13. Remove tray of slides from gas chamber.  Use a razor blade to cleanly remove cover-slips, and place the slides into a dH20 filled plastic Coplin jar.

 

14. To wash off excess monomer, allow slides to incubate in dH20 for 30 minutes with slow shaking.

 

15. Remove slides from Coplin jar and place face up in PCR hood.

 

16. Allow slides to dry for 30 minutes under hood (no more!)

 

 

REAGENT DIFFUSE-IN

 

1. While the slides are drying, prepare the following diffuse-in mix (250 uL total volume = enough for nine slides)

 

12.5

5 mM dNTPs

25

10x Taq buffer

1.65

30% BSA

2.5

10% Tween

1.25

200 uM RGMP1-F Forward Primer

16.75

Jumpstart TAQ (50,000 Units / uL)

190.35

dH20

 

 

 

2. Apply 25 uL directly to center of gel.  Assuming gel is newly dried, the liquid will ball-up rather than spreading over the surface.

 

3. Apply 18 x 30 mm cover-slip.  Don’t slide it on- drop it on.  The liquid should spread to evenly cover the surface of the gel.

 

4. Apply a SecureSeal chamber.  Seal down edges with blunt end of tweezers. 

 

5. Fill with mineral oil.  We have generally found that ~515 uL of mineral oil will fill the chamber with a little room to spare.  Seal with stickies.

 

 

POLONY PCR AMPLIFICATION

 

Place slides in thermocycler (labels facing out)

 

To PCR, cycle slides as follows:

 

(1)       94 ºC              2 minutes

 

(2)       94 ºC              30 seconds

 

(3)       62 ºC              45 seconds

 

(4)       72 ºC              90 seconds

 

(5)       Go to (2) 43x 

 

(6)       72 ºC              2 minutes

 

(7)       2 ºC                 (forever)

 

NOTE: Both the annealing temperature and the extension time can be adjusted to optimize for the set of amplification primers being used and the length of the PCR products being formed, respectively.

 

 

SLIDE CLEAN UP

 

1. Fill a GLASS Coplin jar with Hexane

 

2. Fill a PLASTIC Coplin jar with Wash 1E

 

3. Without removing cover-slips, place slides in hexane-filled glass Coplin jar & leave for ~5 minutes.  If a cover-slips accidentally comes off, it is not the end of the world.  The gel will be fine.

 

4. Remove slides, cleanly flip off cover-slips, and transfer to Wash 1E-filled Coplin jar for rinse.  While transferring, be sure to remove all residual adhesive from cover chamber with razor blade or fingers.

 

5. Let sit in Wash 1E.  Gels are fine in Wash 1E and can remain indefinitely before proceeding.  Be sure to cap plastic Coplin jar containing slides to prevent evaporation.

 

 

C. Genotyping

 

Single base-extensions (SBE) provide the basis for genotyping, haplotyping, and exon-typing with polonies (the latter two are essentially just multiplexed versions of the first).  Multi-base sequencing (at least in its current implementation) uses a different set of nucleotides and a somewhat different protocol.  SBE can essentially be performed in four steps:

 

-         DENATURATION

-         ANNEALING

-         EXTENSION

-         SCANNING

 

 

DENATURATION

 

Currently the PCR product is present in the polonies in double-stranded form.  Denaturation will permit us to wash out one strand, such that only the strand that contains the acrydite-modified reverse primer will remain covalently attached to the acrylamide gel.

 

1.      Prepare the following 70% formamide denaturation solution (enough for one Coplin jar of slides)

 

  2 mL       20x SSC

     28  mL       Formamide

10 mL       dH20

 

2.      Pour mix into a plastic Coplin jar and heat without lid to 70 degrees in the microwave (use thermometer to periodically track).  Typically only 30 seconds or so is required to heat the formamide to this temperature. 

 

3.      Deposit slides into heated formamide solution, and incubate for 15 minutes with shaking in the 70 degree incubator.

 

4.      Remove jar from incubator and transfer slides to separate plastic Coplin jar containing dH20.

 

5.      Wash slides 1 x 3’ in dH20 on shaker.

 

6.      Wash slides 2 x 4’ in Wash 1E on shaker.

 

ANNEALING

 

In this step, the sequencing primer is annealed to a specific location within the single-stranded PCR product.  The sequencing primer is therefore in the same orientation as the forward amplification primer.  Although it is possible to use the forward amplification primer itself as a sequencing primer, it is generally advisable to use a sequencing primer that is internal to the PCR product (largely to avoid incidental sequencing of spurious PCR products, primer-dimer, etc.)

 

1. The formula for annealing mix (1.2 mL) for any given sequencing primer is:

 

1194 uL          6x SSPE w. Triton X-100

       6 uL          100 uM sequencing primer

 

We have been generally storing annealing mix at room temperature and have found that it does not need to be prepared fresh for each experiment.  Storing at –20 C is possible but can result in precipitation of salts out of solution.

 

2. Perform the following for each slide:

 

Remove from Wash 1E-filled Coplin jar

 

Dry edges of slide with ChemWipe

 

Apply the FrameSeal green chamber base to the slide

 

Use blunt end of tweezers to seal down chamber

 

Add 125 uL of annealing mix to center of each gel

 

Remove plastic layer covering sticky surface of chamber base

 

Cover chamber with plastic seal (NO COVER SLIP, NO MINERAL OIL)

 

Use blunt end of tweezers to seal down chamber & cover

 

3.      Place slides in slide-cycling PCR machine (labels facing out)

 

4.      Run the annealing reaction (Program = Anneal56)

 

a.      94 degrees 6 minutes

b.      56 degrees forever

 

5.      When the 15 minutes are up (e.g. no four degree step), use tweezers to QUICKLY pull off the FrameSeal chambers and transfer slides directly to black wash box filled with Wash 1E.  The goal here is to dilute away the sequencing primer before the temperature can drop (which would permit non-specific binding)

 

6.      Wash slides 2 x  4’ in Wash 1E on shaker.

 

EXTENSION

 

Obviously the selection of specific Cy-label and dNTP combinations will depend on exactly what you are trying to sequence in a specific experiment.  Note that Cy-labeled dNTPs are NOT terminators of polymerization.  More than one labeled or unlabeled dNTP can be included in an extension reaction.  Simply add it to the below formula and reduce the amount of Klenow Extension Buffer accordingly.  In a typical genotyping reaction, we will include two dNTPs, one Cy3 labeled and one Cy5 labeled.  Note that the signal-to-noise ratio for Cy5 is considerably better than Cy3.  Lately, we have taken to adding a single Cy5-labeled base, scanning the gel, and then performing a second extension reaction with the other base, also Cy5 labeled (so that we can avoid using Cy3 and get great signal-to-noise ratios), and then scanning a second time.  The separate can be easily aligned and processed after the fact.

 

(1)   Make the EXTENSION MIXES (should be at ROOM TEMPERATURE).  One needs 45 uL of extension mix per slide.  The forumula for 100 uL of any given extension mix with a single dNTP is as follows:

 

 

0.5 uL                         Klenow (50,000 U / mL)

1.25 uL                       SSB

0.5 uL                         100 uM Cy? dNTP

97.75 uL                     1x Klenow Extension Buffer

 

 

(2)   Equilibrate slides in 1x Klenow buffer (fill plastic Coplin jar with 1x Klenow buffer and transfer the slides to it a few minutes before beginning extension reactions)

 

(3)   Make sure edges of slide are dry prior to adding extension mix (keeps mix on the gel itself)

 

(4)   Add 45 uL of extension mix to each slide.

 

(5)   Allow reaction to go forward for 2 minutes.  Tilt slide around a bit to get extension mix to cover surface of gel

 

(6)   Wash slides 2 x 4’ in Wash 1E

 

 

SCANNING

 

[UNDER CONSTRUCTION]

 

 

D. Haplotyping

 

[UNDER CONSTRUCTION]

 

 

E. Exon Typing

 

[UNDER CONSTRUCTION]

 

 

F. Sequencing

 

Sequencing essentially consists of a series of base additions (e.g. C A T G C A T G C A T G…), interrupted by scanning for data-acquisition, and cleavage for signal-clearing.  In short, the protocol is as follows:

 

Preparatory Steps

 

(1) Perform Polony PCR amplification (as above)

(2) Denature dsDNA (as above)

(3) Anneal sequencing primer (as above)

(4)  Prepare BME-Bleaching Buffer Mix, Iodoacetamide-Alkylation Buffer Mix (special mix protocols below)

(5) Load Klenow polymerase to gel (Klenow loading protocol below)

 

Serial Steps

 

(6) Extend with Cy5-SS-dNTP (base addition protocol below)

(7) Scan slides to acquire data on base addition

(8) Cleave to eliminate signal (cleavage protocol below)

(9) Scan slides to confirm efficient cleavage

(10) Go to Step 6

 

SPECIAL MIX PROTOCOLS

 

BME-Bleaching Buffer Mix = 200 ml 1x Bleaching buffer + 6.8 ml 2-mercaptoethanol

 

Iodoacetamide-Alkylation Buffer Mix = 1300ul Alk buffer + 10mg iodoacetamide.

 

Klenow Polymerase Mix (50 uL per slide) = 48.75 uL 1x Klenow Buffer + 0.5 uL Klenow exo- (50K U/mL) + 0.75 uL SSB

 

Nucleotide Mixes (50 uL per slide per addition) = 48.75 uL 1x Klenow Buffer + 0.5 uL Cy5-SS-dNTP (100 uM) + 0.75 uL SSB

 

KLENOW LOADING PROTOCOL

 

The purpose of this step is essentially to load the Klenow polymerase on to the primed template.  Due to acrylamide-induced reduction of the off-rate, the polymerase should be stable for at least 1 day in Wash 1E at 4 degrees C.  However, we have yet to seriously quantitate the off-rate, and generally proceed to sequencing immediately after Klenow loading.

 

1.         Presumably slides have been PCR amplified & denatured, a sequencing primer has been annealed, and the slides are in Wash 1E

 

2.         Equilibrate slides in 1x Klenow buffer (fill plastic Coplin jar with 1x Klenow buffer and transfer the slides to it a few minutes before beginning extension reactions)

 

3.         Make sure edges of slide are dry prior to adding extension mix (keeps mix on the gel itself)

 

4.         Add 45 uL of Klenow Polymerase Mix to each slide.

 

5.         Allow reaction to go forward for 4 minutes.  Tilt slide around a bit to get extension mix to cover surface of gel

 

6.         Wash slides 2 x 4’ in Wash 1E

 

BASE ADDITION PROTOCOL

 

1.         Equilibrate slides by dipping in 1x Klenow Extension Buffer

 

2.         Add 45 uL of nucleotide mix directly to gel surface and incubate for one minute with gentle tilting

 

3.         Quench reaction by dipping in Quenching Buffer

 

4.         Wash (2 x 4’) in Wash 1E

 

CLEAVING PROTOCOL

 

1. Incubate slides in BME-Bleaching Buffer mix (1 x 10’)

 

2. Wash slides (7 x 30”) in Wash 1E with inversions

 

3. Apply SecureSeal chamber to each slide

 

4. Add ~600 uL of Iodoacetamide-Alkylation Buffer mix and incubate in dark for 5 minutes

 

5. Remove Iodoacetamide-Alkylation Buffer mix and discard in designated waste container

 

6. Wash with ~600 uL of straight Alkylation Buffer by pipetting in and out several times

 

7. Wash (2 x 4’) in Wash 1E

 

 

IV.       APPENDICIES

 

 

A. PRIMER DESIGN

 

See Supplementary Information in Reference 2 for more information on parameters to use with the Primer3 primer picking software.

 

Here are two sets of polony PCR amplification primers that have been particularly successful:

 

Set 1

           

mCD44_a98F (5’ acrydite-modified):    5’-ACACGCTACAGCAAGAAGGGCGAGTAT-3’           

mCD44_863R (unmodified):                    5’-TGGTCTGGGGTCTCTGATGGTTCCT-3’

 

Set 2

CPR (5’ acrydite-modified)                       5’-GCCCGGTCTCGAGCGTCTGTTTA-3’

                        CPF (unmodified):                                     5’-GGGCGGAAGCTTGAAGGAGGTATT-3’

 

B. SOFTWARE

 

This information is contained in a separate document that can be linked to here: Polony Software Manual

 

 

V.        REFERENCES

 

 

1.         Mitra, R and Church, GM (1999) In situ localized amplification and contact replication of many individual DNA molecules.  Nucleic Acids Res. 27(24):e34; pp.1-6.

 

2.         Mitra, RD, Butty, V, Shendure, J, Williams, BR, Housman, DE, and Church, GM (2002) Digital Genotyping and Haplotyping with Polymerase Colonies. (submitted to PNAS, 10-21-02)

 

3.         Mitra RD, Shendure J, Olejnik, J, Church GM (2002) Fluorescent in situ Sequencing on Polymerase Colonies (manuscript in preparation)