Cytoskeleton Methods and Protocols

263 Pages·2001·1.741 MB·English

by  Jorge A. Garcés, Ray H. Gavin (auth.), Ray H. Gavin (eds.)

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Inverse PCR 3
1
Using an Inverse PCR Strategy to Clone Large,
Contiguous Genomic DNA Fragments
Jorge A. Garcés and Ray H. Gavin
1. Introduction
Conventional PCR screens of genomic DNA will often yield a substantial
fragment of the gene of interest. However, identification of flanking sequences
on either side of a known sequence can be problematic with conventional PCR,
in which primers extend a complementary chain in a 5' → 3' direction. How-
ever, if the template DNA is circularized and then used with primers that are
oriented with their 3' ends directed away from each other, amplification around
the circular template results in a linear PCR product consisting of
uncharacterized DNA fragments flanked by the known DNA sequences (Fig. 1).
This variation of the conventional PCR strategy is known as inverse PCR (1,2),
and we have used the technique to amplify myosin sequences in Tetrahymena
(3). In this chapter we describe protocols that enable the investigator to apply
the inverse PCR technique to clone contiguous sequences upstream and down-
stream of a known DNA sequence.
1.1. General Scheme for Cloning Contiguous Sequences
1. Purify and cut genomic DNA sample with appropriate restriction enzyme. EcoR1
is shown in Fig. 1. Black box shows region of initially known sequence.
2. Perform inverse PCR reactions as described using primer sequences within the
region of known sequence.
3. After the inverse PCR product has been cloned and sequenced, a full-length con-
tiguous fragment may be amplified by conventional PCR using primer sequences
at or adjacent to the EcoR1 sites from a genomic DNA template. Search for new
restriction sites within this latest cloned stretch of DNA. A Hind III site is shown
inFig. 1.
From: Methods in Molecular Biology, vol. 161: Cytoskeleton Methods and Protocols
Edited by: R. H. Gavin © Humana Press Inc., Totowa, NJ
3
4 Garcés and Gavin
Fig. 1. General scheme for cloning contiguous sequences.
Inverse PCR 5
4. Cut genomic DNA sample with the second restriction enzyme. This creates two
fragments that will serve as template for the inverse PCR reaction and facilitates
amplification of novel sequences both upstream and downstream of the known
sequence.
5. Obtain additional clones by inverse PCR. The overlap between the new and old
clones allows for proper orientation of the inverse PCR fragments and prevents
the creation of gaps in a contiguous sequence. PCR primers are designed from
regions of known sequence as depicted in the diagram.
6. Amplify full-length contiguous DNA fragment by conventional PCR using
primer sequences at or adjacent to the outermost Hind III sites. Using this strat-
egy, between 5 and 10kb of novel contiguous DNA sequence information can be
obtained after only two rounds of inverse PCR. Further inverse PCR cycles using
fragments left and right of the starting fragment will yield more sequence. This
strategy may be repeated to “walk” both upstream and downstream of a known
DNA sequence.
2. Materials
1. Chroma-Spin column (Clontech) with an exclusion limit of 1000 bp.
2. T4 Ligase.
3. Ligase Buffer: 66 mMTris-HCl (pH 7.6), 6.6 mM MgCl , 0.1 mMATP, 0.1 mM
2
spermidine, 10 mM DTT and stabilizers.
4. PCR Buffer: 25 mMTricine, pH 8.7, 85 mMKOAc, 8–10% glycerol, 2% DMSO,
dNTP mix: 10 mMdNTP mix [200 µMfinal concentration for each dNTP].
5. rTth polymerase-XL (Perkin-Elmer). (seeNote 1).
6. Glycerol reagent.
7. Dimethylsulfoxide (DMSO) reagent.
8. Agarose gel electrophoresis reagents.
9. Clean, sharp razor blades.
3. Methods
3.1. Designing PCR Primers
Design primers suitable for your application (seeNote2).
3.2. Digestion and Purification of Template DNA
1. Mix the following components in a microfuge tube:
a. 30 µg genomic DNA.
b. 30 units of a suitable restriction enzyme (seeNote3).
c. Enzyme buffer according to supplier’s directions.
d. Distilled water to 50 µL.
2. Incubate at 37°C for 1 h.
3. Purify digestion products from step 2 by using a Chroma-Spin column. (see
Note4).
6 Garcés and Gavin
3.3. Circularizing the DNA
1. Make serial dilutions of the purified DNA prepared in Subheading 3.2.
2. Set-up the ligation reaction in small PCR tubes as follows (seeNotes 5 and 6):
a. Diluted DNA.
b. 6 Weiss units of T4 ligase in ligation buffer.
c. Incubate at 16°C for 60 min. (seeNote 7).
3. Purify circularized fragments by using a Chromaspin column.
4. Use the purified, circularized DNA for inverse PCR as described in Sub-
heading 3.4.
3.4. Inverse PCR
3.4.1. DNA Template
Make serial dilutions of the circularized DNA prepared in Subheading 3.3.
3.4.2. PCR Reaction Mixtures (seeNotes 8–10)
3.4.2.1. LOWERLAYER OF PCR MIXTURE
1. Add the following reagents to a PCR tube:
a. 14µL distilled water
b. 12 µL PCR buffer
c. 1 µL primers [0.5 µMeach]
d. 8 µL of dNTP mix
e. 5.0 µL Mg (OAc)2 = 1.25 mMfinal Mg concentration.
2. Place an AmpliWax bead on top of the mixture.
3. Heat the tube at 80°C for 5 min.
4. Resolidify the wax by returning the tube to room T for 5 min.
m
3.4.2.2. UPPER LAYER PCR MIXTURE
Add the following reagents to the PCR tube:
1. 18 µL PCR buffer.
2. 1 µg circularized DNA.
3. 20 µL of rTth polymerase-XL.
4. Distilled water to total a 60 µL volume.
3.4.2.3. PCR CYCLE
Select suitable cycling parameters and initiate the reaction (seeNote 11).
3.4.3. Analyzing the PCR Products
1. Analyze the PCR products using agarose gel electrophoresis.
2. Excise the band of interest and clone into a suitable vector for sequencing and
expression.
Inverse PCR 7
4. Notes
1. This approach has been successfully used to amplify Tetrahymena DNA frag-
ments ranging from 2–8 kb. It is important to use a cocktail mixture of DNA
polymerase enzymes such as rTth polymerase-XL (Perkin-Elmer) or Taq/pfu
(Stratagene), which are proportionally premixed to enhance amplification of long
PCR products and allow for proofreading activity to minimize random mutagen-
esis of amplification products.
2. For amplification of ciliated protozoan sequences, recommended characteristics
for primers include: oligomers consisting of 21–24 nucleotides, a T between
m
60–68°C, and 50% G-C rich composition. The T of the primers should not dif-
m
fer by more than 3–5°from each other. If the T of the primers varies by 5–10°,
m
use a touch down approach from the highest to the lowest T value. For example,
m
in a two-step PCR reaction in which the T of one primer is 68°and the T of the
m m
second primer is 62°, start with a cycle consisting of a 94-degree denaturation
step followed by a 68 degree combined annealing/extension step. Subtract 0.5°C
from the annealing/extension step in each subsequent PCR cycle. A 5 s time
increment should be added to the annealing/extension step of each subsequent
cycle to compensate for a slight decrease in the DNA polymerase’s rate of nucle-
otide incorporation as the reaction progresses. The last eighteen of thirty cycles
consist of a 94-degree denaturation step followed by a 62 degree combined
annealing/extension step.
3. A suitable restriction enzyme should generate fragments about 2–3 kb (seeNote5)
with a four base overhang to facilitate ligation. Select an enzyme that has a recog-
nition site compatible with the template DNA based on G-C/A-T content. For
example, in Tetrahymena, A-T content of genomic DNA is high, and enzymes
such as EcoRl and Hind III, which cut at A-T rich sites, yield fragments in the
desired size range. In contrast, enzymes such as BamH1, which cuts at G-C rich
sites, yield fragments that are too long for efficient use in inverse PCR. Hybridiza-
tion blotting can be used to confirm the identity of the restriction fragments.
4. Alternatively, gel electrophoresis can be used to purify the digestion products
and to identify components of optimum size for circularization (seeNote 5). Use
a sharp clean razor blade to excise the band of interest from the gel and purify
fragments using the Gel Extraction Kit (Qiagen).
5. Many of the problems encountered with inverse PCR can often be traced to char-
acteristics of the template DNA. In creating the circular DNA template, there is
competition between concatamer formation and circularization of DNA frag-
ments. The optimum DNA concentration that promotes circularization varies
with the length of the DNA to be circularized and must be determined empiri-
cally for each template. In general, the optimum size range for efficient circular-
ization is 2–3 kb. Fragments outside this range fail to circularize efficiently.
6. Perform a range of ligation reactions with varying concentrations (10 ng–1.0 µg)
of the purified, digested DNA.
7. A 1 h incubation period is used instead of the standard overnight incubation
because it appears optimal for biasing circularization over concatamer formation.
8 Garcés and Gavin
8. Use a hot start approach to minimize mispriming. For hot start PCR, a wax bead
(AmpliWax PCR Gem 100; Perkin Elmer) is used as a barrier between layers
consisting of different PCR components. Melting and subsequent solidification
of the wax bead allows PCR components to be mixed at a T higher than the
m
highest annealing T in the PCR cycle. The wax also eliminates the need for oil or
m
a heated lid to minimize condensation at the top of the tube.
9. In order to lower the high melting temperatures required for denaturation of
closed circular DNA, the DNA can be linearized at a site between primers. Alter-
natively, a PCR buffer containing DMSO and glycerol as solvents can be used to
overcome difficult secondary structure restrictions.
10. Magnesium concentration can be varied as desired.
11. A recommended cycle consists of a pre-PCR hold at 94°for 10–15 s and 12 rounds
of a two step PCR-cycling program that combines annealing and extension steps.
Each cycle consist of a denaturation step at 94° for 12 s, an annealing/extension
step at 65° for 5–12 min (1 kb/min), and 18 cycles of the same two-step process
with time increments of 12 s per cycle added to the annealing/extension step.
An extension at 72° for 10–15 min is used as the final step in the reaction.
Acknowledgments
This work was supported by Grant MCB 9808301 from the National Sci-
ence Foundation to RHG.
References
1. Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an
inverse polymerase chain reaction. Genetics120, 621–625.
2. Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) A procedure for in vitro
amplification of DNA segments that lie outside the boundaries of known
sequences.Nucleic AcidsRes.16, 8186.
3. Garcés, J. and Gavin, R. H. (1998) A PCR screen identifies a novel, unconven-
tional myosin heavy chain gene (MYO1) in Tetrahymena thermophila. J. Euk.
Microbiol.45, 252–259.
Microsequencing of Myosins 9
2
Microsequencing of Myosins for PCR Primer Design
Elaine L. Bearer
1. Introduction
1.1. Background
Their large size and their relative resistance to proteolytic cleavage (1)make
myosins particularly difficult substrates for the acquisition of their peptide
sequences by standard protocols. For this reason, instead of identifying
myosins first according to their biochemical activity and then obtaining their
sequences, PCR and other DNA-based techniques exploiting the highly con-
served sequences in the amino end, the “head” domain, have been used to find
new myosins (2–5). However, such identification of myosins by sequence
leaves open the question of their function. If peptide sequence could be
obtained from myosin proteins whose biochemical behavior was known, then
the gap between function and sequence could be bridged. We describe here a
method that enabled us to acquire peptide sequences of semi-purified myosins (6).
1.2. Rationale for the Strategy
There are several difficulties that must be overcome to obtain peptide
sequences from myosins. First, a sufficient amount of protein that is reason-
ably pure must be obtained. For muscle and non-muscle myosin IIs this is rarely
a problem, although it can pose problems if the organism or the tissue to be
studied is small, rare or otherwise difficult to obtain in large quantities—as is
the case for axoplasm from the squid giant axon (6–10) or the organism
Tetrahymena (2). This chapter will not address problems of abundance since
they pertain to individual applications.
Once sufficient amounts (micrograms) of a myosin have been obtained,
the protein must be proteolytically cleaved into peptide fragments. These
fragments must then be purified to homogeneity for Edman digestion to
From: Methods in Molecular Biology, vol. 161: Cytoskeleton Methods and Protocols
Edited by: R. H. Gavin © Humana Press Inc., Totowa, NJ
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10 Bearer
produce readable sequence. Myosin is relatively protease resistant. The coil-
coil region in the tail and the tight packing of the amino head domain prevent
cleavage at all but the neck region that links the two domains. Chymotrypsin,
trypsin and pronase all produce cleavage in the neck and leave the rest of the
molecule intact even under conditions when other proteins would be reduced
to peptide fragments. Since tight packing of these domains in myosin is likely
responsible for this protease resistance, we unwind the myosin before pro-
teolysis with sodium dodecyl sulfate (SDS),although other chaotropic agents
may work as well. While low levels of SDS unwind myosin, the proteolytic
enzyme endolys C, a serine protease, remains active in SDS. This strategy
results in sensitivity to proteolysis throughout the myosin molecule.
After proteolytic cleavage, the resultant peptide fragments must be sepa-
rated. For a large molecule this is particularly difficult as complete diges-
tion results in a very large number of small peptides, often with very similar
properties making them difficult to separate. Hence, chromatographic sepa-
ration must be performed in two steps in order to obtain adequately pure
peptides. Several factors including inadequate amount of starting material,
incomplete digestion of proteins, or loss of peptides during the chromato-
graphic separations, can lead to insufficient quantities of each purified pep-
tide for the final sequencing reaction.
The following protocol was first developed using squid muscle myosin as
starting material. At the time, that myosin had not been purified, and its
sequence had not been obtained. The sequence of squid muscle myosin II
was ultimately obtained by a combination of two approaches: (1) PCR sequenc-
ingusing primers to hypothetically homologous domains from scallop muscle
myosin, a myosin from a related species, and (2) PCR sequencing using degen-
erate PCR primers based on the peptide sequences we obtained from the puri-
fied squid protein (11,12). After devising a strategy applicable to muscle
myosins, we then applied this strategy to the identification of a squid brain
myosin not so easily obtained in large amounts (6,10,13,14). Others have since
applied our strategy to obtain sequences from other squid optic lobe myosins
(15). This strategy is therefore likely to be of use for most myosins even those
that are significantly less abundant than muscle myosin. This method can be
applied to purified myosin in solution or semi-pure myosin excised from a
Coomassie-stained SDS-PAGE gel. The strategy as we applied it was a compi-
lation and modification of previously reported methods (16–18).
2. Materials
2.1. Reagents and Glassware
1. Endoproteinase Lys-C (Wako Chemicals, Richmond, VA).
2. Guanadinium hydrochloride (Sigma Chemicals, St. Louis, MO).
Microsequencing of Myosins 11
3. Biospin-30 chromatography column (BioRad, Hercules, CA).
4. Millex HV filter (Millipore, Bedford, MA).
5. Vydac 218TP52 column (Separations Industires, Metuchen, NJ).
6. YMC ODS AQ column (YMC, Willmington, NC).
7. Briobrene-treated glass fiber filter (Applied Biosystems, Foster City, CA).
2.2. Buffers
1. Myosin storage buffer (MS): 0.5MKCl, 3 mMNaN , 2 mMMgCl , 1 mMDTT,
3 2
2 mM EGTA, 4 mM NaHCO (pH 7.0), 50% glycerol.
3
2. NED: 0.1 mM NaHCO , 0.1 mM EGTA, 0.1 mM DTT.
3
3. Digestion buffer (DB): 0.1 % SDS, 100 mM NH HCO
4 3.
4. Solvent A: 0.1% trifluoroacetic acid in water.
5. Solvent B: 0.1% trifluoroacetic acid in acetonitrile.
2.3. Equipment
1. Beckman System Gold HPLC equipped with an autosampler (model 507), diode
array detector (model 168), and programmable solvent module (model 126).
2. Applied Biosystems pulsed-liquid protein sequencer (model 477A) equipped
with PTH analyzer (model 120A).
3. Applied Biosystems data analysis system (model 610A).
3. Methods
3.1. Proteolytic Digestion of Myosins (see Notes 1–4)
1. Precipitate purified myosin out of buffer (MS) by adding a tenfold volume of
NED to 40 µg of the myosin suspension.
2. Incubate for 1–4 h on ice.
3. Collect the precipitate by centrifugation at 16,000g in a microfuge for 30 min
(seeNote 1).
4. While the myosin is precipitating, equilibrate the BioSpin 30 column. First
remove the packing buffer by centrifuging in the 2 mL tube (comes with the kit
from BioRad) for 1 min at 1,000gor until the entire amount has passed through
the column. Discard the buffer. Next add 100 µL of DB to the top of the column
and centrifuge again. It is best to use a swinging bucket rotor, but you can also do
this with a benchtop microfuge. After the DB has passed through the column,
remove it, and let the tube sit on the bench until loading.
5. Resuspend the myosin precipitate in 80 µL of DB. Heat to boiling (95–100°C)
for 5 min.
6. Apply 80 µL of denatured protein solution to the DB-equilibrated BioSpin 30 col-
umn and centrifuge the tube for 4 min at 1000g. This removes light chains that are
released during the denaturation process, and any other contaminating proteins
smaller than 40 kDa which are retained in the column. Dispose of the column,
and use the solution that passed through it for subsequent steps.
7. Add to the column eluate 6 µL of DB containing 1.5 µg of endoproteinase Lys C.
Put samples in 37°C water bath and allow to digest overnight (16 h).