ABRF '96: Biomolecular Techniques

Tutorial #4

1. Factors that affect the quality of sequencing.
2. Types of difficult templates.
3. Sequencing PCR products.

• Purity
• Host strains
• Contaminants
• Quantity
• Primer quality

Purity of DNA - use robust template preparations. Recommended in order of reliability:

Qiagen tip 20, tip100

Alkali lysis/PEG precipitaion

CsCl banding

Wizard, 5' 3', SNAP columns.

Classical alkali lysis

Host strains

DH5alpha and HB101 work well

Common contaminants

EDTA at concentrations > 0.5mM - compromises Mg concentration.

Phenol - quenches fluorescent dyes.

High salts - inhibits enzyme.

SOLUTIONS:

• O.D. template - this can save a lot of frustration for a minimal investment of time.
• Clean up template. e.g.
  1. C100, microcon 100, Quiaquick.
  2. Ethanol precipitation.
  3. PEG precipitation - add 0.16 volumes of 5M NaCl and 1 total volume of 13% PEG. Incubate on ice for 20 minutes. Rinse with 70% ethanol and resuspend in water.
• Slide 1: O.D. trace from 310 to 220 showing DNA in water.
• Slide 2: O.D. trace from 310 to 220 showing DNA in EDTA - EDTA absorbs at A220/240
• Slide 3: O.D. trace from 310 to 220 showing DNA with phenol contamination - phenol absorbs at A270.

DNA QUANTITY

• Slide 4: Too much template will give a top heavy appearance and you may see off scale peaks and/or trailing peaks.
• Slide 5: Too little template will cause low signal and noise to the extent that bases cannot be called.

Taq FS requires less template and seems to tolerate a larger window of template concentration.

5ng/100 bases has been suggested by some as general guidelines for PCR products.

PRIMER QUALITY

Primers should have most of the following characteristics to be able to produce good, consistent sequencing data.

• High purity
• No mismatches
• No potential alternative binding site in the template
• No secondary structures present, especially at the 3' end
• 18 bases minimum, longer for AT rich primers
• Tm of around 55-60 degrees - if not make the primer longer
• Avoid runs of more than 4 of the same bases
• Slide 6: Noisy primer showing multiple peaks caused by random priming of a N-1 primer synthesis.

2. TYPES OF DIFFICULT TEMPLATES.

• High G/C content
• AT rich sequences
• Secondary Structure
• Repeats
• Homopolymer regions
• Cosmids, P1 clones

Be prepared to use all possible sequencing chemistries.
No SINGLE chemistry will work on all templates, although Taq FS does a very good job on most difficult templates.

High G/C Content

• >60% GC content may be difficult to sequence.
• Dye terminator performs better than dye primer.
• Easiest modification is to add 5% DMSO final concentration to the reaction mix - usually very successful! 10% DMSO is too high - tends to kill the reaction. Some labs use 5% DMSO routinely in all terminator reactions with no adverse effect. Sometimes, you have to repeat a reaction which turns out to be very A-T rich, but this happens rarely in my experience.
• Sequence the opposite strand to help resolve ambiguities.
• Try a combination of the following modifications:
  • Raise denaturing temperature to 98 degrees
  • Add more Taq
  • Increase cycles to 30 for terminators
  • Use 2x dNTPs to balance out top heavy reactions
  • Try a pre-denaturation step at 96 degrees for 4-5 minutes
  • Slide 7: Shows a sequence of 85% G/C over 200 bases, sequenced with Taq FS containing 5% DMSO, standard ABI cycling conditions.
  • Slide 8: in the The upper panel shows G/C compressions around bases 50-60 and 119-121 in a dye primer sequence . The lower panel shows the same template sequenced with Taq dye terminators.

  AT rich sequences

  • Generally not as problematic as G/C rich templates.
  • If a primer is AT rich, increase the length of the primer to about 24-26 bases to increase the melting temperature closer to 55 degrees.
  • Try dye primer sequencing if appropriate, as dye primers sequence more evenly through AT regions.
  • If all else fails, try Sequenase dye terminators. However the chemistry is not very robust and 5ug of template is usually required. A five filter wheel is also required to analyze the dye set used for Sequenase on the ABI system.

  Secondary Structure -
  Inverted repeats, Palindromes

  An abrubt loss of signal usually signifies a DNA sequence structure problem, due to the inability of the enzyme to proceed through the problem area.

  • 5% DMSO sometimes helps
  • Choose any of the combinations suggested for G-C rich template
  • Try sequenase dye terminators

  Repeats

  • Repeat sequences such as di,tri and tetra-nucleotide repeats usually are not problematic in cloned material. In PCR products, though, enzyme slippage occurs and sequence cannot be obtained through the repeat region. Ref: Odelberg et al, Nucleic Acids Research.1995, Vol. 23, No.11, 2049-2057
  • Longer repeat sequences such as variable tandem repeats of 30 or more bases repeated many times are difficult to deal with. Run LongRanger gels on 48 cm plates for the longest read possible or perform well controlled directed deletions. E.g., ExoIII deletion series on the template.
  • AG repeat sequences can be problematic because Taq FS produces a weak G signal after A in terminator data. Try using more template or sequencing the opposite strand. Use dye primers if applicable. Use sequenase dye terminators as a last resort.
  • Slide 9: Shows a 24 base pair stretch of sequence (highlighted) which is repeated over 30 times in the sequence.
  • Slide 10: Shows G after A problems with Taq CS and Taq FS. Often sequencing the opposite strand resolves these problems.

  Homopolymer regions

  • Problems can arise when more than 10 of the same bases occur consecutively or sometimes less than 10 for G and C.
  • Generally one can sequence through the shorter runs of polyT's, but enzyme slippage can occur when the number of bases is greater than 30. Surrounding sequence may also play a role in the success of sequencing throu gh homopolymer regions.
  • Poly T solutions: Karl Hager uses an anchored primer of 25 T residues with a degenerate 3' base of G, C, and A. This is a useful tool to help sequence through cDNA clones.
  • Poly G and Poly C sequences seem to be the most difficult for many people. Karl Hager suggests making a primer that will anneal 20 bases upstream from the homopolymer to force the polymerase through the problem. If all else fails, try Exo III deletions on the clone. It is important to use an exonuclease deficient enzyme for sequencing through homopolymer regions. E.g., TAQ FS
  • Slide 11: Shows a poly T track of less than 30 bases which can be sequenced through, but notice the increase in background noise after the poly T sequence.
  • Slide 12: Shows a very long poly A track which proved impossible to sequence through. Use an anchored primer in cases like this to try to force the enzyme to extend through this difficult region.
  • Slide 13: Shows a poly A region in a PCR product. It is impossible to sequence through this region. Slippage during PCR cycles causes the sequence after the poly A to become unreadable, notice the "wave" form. Sequence from the opposite end to complete the sequence.

  Cosmid and P1 clones

  • Purity of the preparation is highly critical for success. Protocols provided below.
  • Grow fresh colonies and sequence freshly prepared DNA within 24 hours if possible.
  • Use only dye terminator chemistry at full volume reactions, i.e. 20ul.
  • Use 1-2 ug DNA - no need to alter primer quantity - use 3.2 pMoles as usual.
  • Slide 14: Cosmid sequence of a fairly G/C rich cosmid, sequenced using the standard ABI dye terminator protocol with a pre-denaturing step at 96 degrees for 30 seconds.
  • Slide 15: Sequence of a P1 clone sequenced with the standard ABI dye terminator protocol with a pre-denaturing step at 96 degrees for 30 seconds.

  ---

  
  COSMID PROTOCOL
  Maurine Hobbs, University of Utah.

  Day One

  1. Start overnight cultures of Cosmid in fresh L-Broth + 25 ug/ml kanamycin. 4 x 250 ml each, inoculate each with metal loop from frozen glycerol stocks.
  2. Grow cultures for 12 hours (do not let go for more than 14 hrs.)

  Day Two

  3. Spin down cultures in 250 ml bottles in Beckman JA-17 rotor at 6,000 rpm for 15 min.
  4. Pour off sups, freeze pellets at -20 degrees C for 10 min.
  5. Resuspend in 10 ml GET solution + 1 mg/ml lysozyme each, transfer 5 ml to each of two 30 ml Oakridge tubes. (8 tubes total).

      

     • GET Solution
       50 mM glucose
       25 mM Tris-Cl (pH8.0)
       10 mM EDTA (pH8.0)
       Make 100 ml batches, autoclave 15 mins, store 4 degrees C.

      

  6. Add 10 ml of SDS/NaOH solution (1% SDS, 0.2N NaOH) to each tube, mix by gentle inversion 10 times. Lyse on ice for 5 min. (Potential denaturation of supercoils if left longer)
  7. Add 7.5 ml 3M NaOAc, pH 4.5 to each tube, mix, on ice 10 min to 1hr.
  8. Spin out bacterial debris in a Beckman JA-20 rotor at 10,000 rpm for 15 min.
  9. Pour supernatants to clean tubes, add 14 ml Isopropanol. Let sit at room temperature for 2 hrs.
  10. Spin down DNA at 10,000 rpm for 15 min. in JA-20. Pour off supernatants, drain on paper towel for 5 -10 min.
  11. Resuspend in 4 ml/tube TE (10mM Tris-HCl, 1mM EDTA) on gentle shaker overnight at room temperature. (Have tubes in a tilted rack so the TE covers the pellet)

  Day Three

  12. Combine all samples, add 34.8 gm CsCl. Mix on shaker until dissolved.
  13. Add 800 ul of 10 mg/ml EtBr. Weigh 1 ml of solution, add CsCl or TE to adjust the weight of the solution to 1.55 to 1.59 g/ml.
  14. Fill Beckman Ultracentrifuge tubes, seal and spin at 70,000 rpm overnight.

  Day Four

  15. Break seal on tubes, pull lower bands seen with UV light, combine into one tube.
  16. Extract EtBr with NaCl saturated Isopropanol, extract two times beyond the loss of pink color in the Isopropanol.
  17. Precipitate the DNA by adding three volumes of H2O, bring up to 0.1M NaCl, and add two volumes of EtOH. (i.e. 1.25ml cosmid, 3.75ml water, 100ul 5mNaCl, and 10ml EtOH.) Put at -20 degrees C overnight.

  Day Five

  18. Spin down DNA in Corex tubes with rubber sleeves at 10,000 rpm in the JA-20 rotor for 15 minutes. Drain off EtOH, invert tubes on paper towels for 5 min.
  19. Resuspend DNA in 200 ul H2O per tube. Combine two tubes each (400 ul), add 8 ul 5M NaCl (0.1M NaCl final conc.), and 1 ml EtOH. On ice for 15 min.
  20. Spin down in microfuge for 10 min., remove EtOH, wash with 70% EtOH, spin down 10 min. in microfuge, remove EtOH, air dry 10 - 20 min. on bench top. Resuspend in 400 ul H2O. Take absorbance readings at 260 and 280 nm. of sample diluted 1:20 to 1:50.

  YIELD:

  • <600ug considered poor
    600ug - 1mg is average.

  ---

  
  MAXI PREP OF P1 DNA
  DORA STAUFFER / DR. MARK LEPPERT
  Dept. of Human Genetics, University of Utah.

   

  DAY 1

  • Inoculate 5 ml LB broth with 50 ul P1 glycerol stock. Grow 5 hours.
  • Inoculate 500 ml of LB containing 25 ug/ml kanamycin with 2-4 ml of the fresh 5 hour P1 culture. Grow 16-18 hours.

  DAY 2

  • Spin cells at 5500 rpm in a 500 ml bottle in JA-10 rotor for 10 min.
  • Discard supernatant and resuspend pellet in 60 ml of GTE buffer (solution 1).
  • Add 0.3g of powdered lysozyme and incubate 30 min at room temp.
  • Add 120 ml alkaline-SDS (solution 2, prepared just prior to use).
  • Mix gently and put on ice for 30 min.
  • Add 40 ml KOAc (solution 3).
  • Mix gently and put on ice for 10 min.
  • Spin at 9000 rpm for 20 min in JA-10 rotor.
  • Strain supernatent through a 2" X 2" gauze pad into a 500 ml centrifuge bottle.
  • Fill bottle with iso-propanol (at least 0.6 vol.). Store at -20 C overnight.

  DAY 3

  • Spin at 10,000 rpm for 5 min.
  • Discard supernatent.
  • Resuspend the pellet in 9.5 ml of TE-3 buffer (10mM Tris-Hcl 7.4, 1mM EDTA) with gentle shaking for 15 min.
  • Transfer to a 50 ml conical polypropylene centrifuge tube.
  • Add 10.5 g of CsCl and mix gently to dissolve.
  • Add 1 ml of ethidium bromide (10 mg/ml).
  • Spin at 3000 rpm for 10 min in J-6 centrifuge.
  • Transfer liquid to a 3" X 5/8" ultracentrifuge tube and seal.
  • Spin 16 - 20 hours at 55,000 rpm. in 70.1 Ti rotor.

  DAY 4

  • Collect lower supercoiled band and transfer to a 15 ml conical centrifuge tube.
  • Extract 5X 2 ml with water-saturated n-Butanol.
  • Add 4 ml water.
  • Transfer to 50 ml.conical centrifuge tube.
  • Fill tube to 45 ml with absolute EtOH and store at -20 C overnight.

  DAY 5

  • Spin at 3500 rpm for 30 min. in J-6 centrifuge.
  • Wash pellet with 1 ml of 70% EtOH.
  • Remove EtOH and air dry pellet.
  • Resuspend pellet in 200 ul of TE-4.
  • Incubation at 37 C aids in dissolvin g the DNA.
  • Add 5 ul RNAse (500ng/ul) and incubate at 37 C for 60 minutes.
  • Extract with phenol-chloroform.
  • Ethanol precipitate.
  • Dissolve pellet in 200 ul TE-4 (10mM Tris 7.4, 0.1mM EDTA)

  Determine concentration by O.D.

  YIELD:	5ug = poor	300-500ug = average
  	Can get up to 1 milligram!

   

  SOLUTION 1

  	GLUCOSE	4.5g
  	1M TRIS-HCL	6.25ML
  	0.5M EDTA	5.0ML
  	WATER TO	250 ML

   

  SOLUTION 2 : PREPARE JUST PRIOR TO USE

  	10% SDS	10ML
  	5N NaOH	4ML
  	WATER TO	100ML

  SOLUTION 3

  	5M KOAc	60ML
  	GL.HOAc	11.5ML
  	WATER TO	100ML

  ---

  3. Sequencing PCR products.

  • Avoids cloning
  • Template can be generated even from degraded DNA (forensic, Histology slides)
  • Can be generated from very limited amount of material
  • Strategies
  • Conditions
  • PCR cleanup
  • What chemistry to use?
  • Sequencing for Heterozygote mutations

  Strategies for PCR Sequencing

  • Single amplification
    • PCR with single set of primers
    • Sequence with the same primers
    • E.g., M13 and colony PCR
  • Nested PCR
    • 2 separate amplifications
    • The second PCR with primers internal to the first pair
    • offers more selectivity, can produce data with less noise
  • Universal tailed primers
    • Custom primers are tailed on their 5' ends with standard M13 primer sequences, so that dye primer chemistry can be used with Taq FS. Useful for Heterozygous mutation detection.
  • Magnetic bead capture of ss DNA from PCR products. One of the PCR primers is 5' biotinylated. PCR is performed with the biotinylated primer at limiting concentration.The resulting PCR forms a stable biotin/streptavidin complex when mixed with Dynal streptavidin beads. The non-biotinylated strand is denatured off the bead and the immobilised strand can be sequenced as single strand DNA.

  ---

  
  

  Conditions for generation of sequencing quality PCR

  GUIDELINES FOR GENERATION
  OF PCR PRODUCTS FOR SEQUENCING

  target DNA	300-50 pg for plasmid/simple targets 200 ng for genomic DNA
  PCR primers	5 pmoles total
  1.25mM dntps	2-16ul
  10x PCR buffer	10ul
  water	q.s. 100ul

  
  

  • HOT START
    • Pre-heat a DNA thermal cycler to 95 degrees.
    • Place tubes in block.
    • Quickly add 2U TAQ polymerase diluted ready for use.
  • Start desired run cycle, x25, linked to soak at 4 degrees.
    • 95 C, 30 secs.
    • 55 C, 30 secs. - depending on Tm (2-3 degrees below Tm)
    • 72 C, 1 min. (P.E. 480)
  • Remove excess primers and nucleotides (c-100, Quiaquick, etc). Check for product on an agarose gel.

  If universal tailed primers are used and dntps are limiting, PCR product may be diluted 1/10 and sequenced with dye primers without further purification.

  PCR cleanup

  • Ultrafiltration, e.g. Centricon 100, microcon 100 ( Amicon )
    • Fast, effective, good recovery. About $2
  • Qiaquick (Qiagen)
    • Fast, effective, good recovery. About $1
  • Exo-Sap digestion (USB)
    • 20ul PCR product
    • 1ul Exonuclease I (10 Units/ul)
    • 1ul Shrimp Alk. Phosphatase (2 Units/ul)
    • incubate at 37 degrees, 30 mins.
    • Inactivate at 80 degrees, 15 mins.
    • reasonably fast, effective, inexpensive at about $0.45
  • Geneclean or Quiaquick gel extraction kit for PCR bands cut out from agarose gels
  • No clean up! When primers and dNTPs are limiting, PCR product may diluted and sequenced with dye primers only.
  • Slide 16: Compares exo sap and Qiaquick for template cleanup. Top panels are untreated PCR product sequenced directly. Bottom left panel is Qiaqick treated then sequenced. Bottom right is exo sap treated then sequenced. All sequencing was Dye terminator cycle sequencing, ABI standard protocol.

  What Chemistry to use?

  PRIMER less rigorous purification even peak heights 4 tube reaction, lower throughput

  TERMINATOR more rigorous purification very uneven peak heights 1 tube reaction, higher throughput

Conclusions:

• For straight forward sequence aquisition, terminators are good.
• For unknown mutation detection the even peak heights produced by dye primers and Taq FS should be used. Sequence both strands!
• For known mutations, dye terminators can be used. You may have to sequence both strands.
• Taq FS has all but eliminated the need to sequence ss DNA from PCR captured on magnetic beads. This strategy could still be useful for PCR products that prove difficult to sequence by the above methods.
• Slide 17: PCR product containing a single base mutation was sequenced using three different sequencing chemistries. The top panel shows single strand PCR immobilised on streptavidin bead complex and sequenced with Sequenase enzyme. Note the very low background and very even peak heights. The middle panel shows Taq FS enzyme and dye primer cycle sequencing on ds PCR product - very similar results to Sequenase. The bottom panel shows taq FS and dye terminator cycle sequencing on ds PCR product. Note the higher background and the consequent difficulty in calling overlapping heterozygous peaks.
• Slide 18: PCR product containing a 2 base pair deletion was sequenced with three different sequencing chemistries. The top panel shows the older Taq CS enzyme with dye primer cycle sequencing. The middle panel shows Taq FS with dye primer cycle sequencing . The botttom panel shows Taq FS with dye terminator cycle sequencing. For insertions and deletions, you can obtain good results with any of the chemistries, but primer sequencing still gives more even peak heights often allowing you to be able to distinguish the actual number of inserted/deleted bases.

Sequencing for heterozygote mutations.

This is probably one of the more demanding DNA sequencing procedures!

• PCR products need to be rigorously optimised to avoid the production of "ghost" bands. These non specific products appear as multiple peaks and can be mistaken for false positives.
• Use dye primer sequencing chemistry and be prepared to sequence both strands. Taq FS and dye primers, produce sequences with very even signals over the 4 bases.
• Insertion and deletion mutations are easy to spot with dye terminator or dye primer chemistry, but for detecting single base changes, dye primer is more sensitive.
• Use a computer program like Sequencher ( Genecodes ) to help search for multiple peaks. Look not only for the appearance of a new allelle, but for the subsequent 50% drop in signal from the normal allelle.
• Slide 19: Too much primer (50pmoles) used in PCR. Note the large peak at 100 bases.
• Slide 20: Optimised primer concentration (5 pmoles). Large peak disappears.
• Slide 21: Long PCR products can cause problems with various stops i.e.generation of non specific PCR products. The longer the PCR product, the higher the chance of producing spurious bands, thus optimization becomes even more important.
• Slide 22: The bottom panel shows "A" noise where the A signal has dropped out due to inadequate addition of DNA or sequencing mix. e.g. C- 250 A-35 G-80 T-50
• Slide 23: An example of a stop causes problems when screening for heterozygous mutations. Could be due to inadequate cleanup of template or enzyme pausing. Sequence the opposite strand and/or make new PCR product.
• Slide 24: Another example of how stops/noise make heterozygote calling very difficult.
• Slide 25: False heterozygote peak. Sequence the opposite strand.
• Slide 26: False heterozygote 2
• Slide 27: Trailing peaks make heterozygote calling difficult. Probably due to gel quality or overlaoding of the gel, especially if the instrument is a 377.
• Slide 28: Demonstrates the use of "Sequencher" -- program in aligning various family members having a mutation in the breast cancer gene.
• Slide 29: Sequencher program used to align the same exon sequence from various patients, facilitates identifying common known and unknown polymorphisms. Use the "mark selection as a feature" to highlight previously reported mutations or polymorphisms.