Ancient DNA: Methods and Protocols (22 page)

PCR inhibitors are often coextracted with aDNA, as samples have often been exposed to environmental contaminants for tens of thousands of years. To minimize inhibition, serum albumin, and commonly bovine serum albumin (BSA), can be included in aDNA PCR. BSA binds PCR-inhibiting coextracts and prevents target DNA from adhering to the tube rather than being amplifi ed.

Including BSA can dramatically impr
ove PCR success ( 3 )
and is useful as a troubleshooting measure when PCRs are unsuccessful.

DNA damage is also common in aDNA extracts. Several measures have been recommended to deal with damage in PCR of

ancient specimens, including pretreatment with uracil DNA glycosylase (UNG or UDG) to remove uracil
( 5 )
or
N
-phenacylthiazolium bromide (PTB) to cleave cr
osslinks

( 6
) . Although treatments

designed to remove uracil can be benefi cial, many aDNA researchers are reassured of the authenticity of the resulting ancient sequences when random C–T (or G–A, if the cloned product is the reverse strand pairing to the strand on which the damage occurred) transitions are observed in cloned products of a PCR, as this form of damage is common in ancient samples.

It is important to note that aDNA PCR will often require

much more optimization than modern DNA PCR because template quantity, quality, and level of inhibition are unique to each extract. The quantity and quality of starting template copies can be highly stochastic even between aliquots of a single DNA extract, 15 PCR

Amplifi cation, Cloning, and Sequencing of Ancient DNA

113

so multiple PCRs from different extracts are suggested to evaluate the consistency of sequencing results and confi rm the consensus sequence. Cloning multiple PCR amplifi cations derived from poorly preserved samples is highly recommended. Cloning only a single PCR product may be misleading if the starting copy is damaged, and a miscoded base is incorporated in an early PCR cycle.

As this book is targeted for specifi cally aDNA research, famil-iarity with basic PCR and routine molecular biology lab protocols, such as running an agarose gel and pipetting, is assumed. We refer readers with no experience with PCR or basic molecular biology methods to more general works
( 7, 8 )
.

2. Methods

All reagents and plastics should be sterile, DNA and DNAse free.

All solutions should be molecular biology grade or similar.

2.1. Required

1. Deoxynucleoside triphosphates (dNTPs) of 100

μ M each,

Reagents

combined in equal volume to yield a dNTP mix of 25 μ M each dATP, dGTP, dTTP, and dCTP.

2. DNA Polymerase + buffer supplied with polymerase (see Note 1).

3. Magnesium ions supplied separately with polymerase usually as MgCl or MgSO .

2

4

4. Forward and reverse primers diluted to 10 μ M each.

5. BSA, rabbit serum albumin (RSA), or a different serum albumin prepared to 10 mg/mL solution in sterile water (see Note 2).

6. Barrier/fi lter tips and PCR reaction tubes/plates.

7. Thermocycler with heated lid.

8. DNA template.

2.2. Agarose Gel

1. 2% agarose gel.

Visualization

2. 50× TAE (500 mL: 121 g Tris, 28.6 mL glacial acetic acid, 50 mL 0.5 M EDTA pH 8.0), diluted to 1× for running

buffer.

3. 6× loading dye (0.25% Orange-G (TCI), 0.1875% xylene

cyanol (IBI Scientifi c), 30% glycerol).

4. DNA ladder. The ladder can be diluted with TE buffer: 125 μ L

(0.25 μ g) prepared ladder + 1,125 μ L TE + 250 μ L 6× loading dye (supplied with ladder).

5. Agarose gel electrophoresis rig and power supply.

6. Ethidium bromide (EtBr) and a UV transilluminator.

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T.L. Fulton and M. Stiller

2.3. PCR Purifi cation

1. A commercial PCR purifi cation kit, e.g., Qiagen, Millipore, ExoSAP, Agencourt AMPure XP.

2.4. BigDye

1. BigDye sequencing kit (Applied Biosystems).

(Applied Biosystems)

2. 1 μ M primer.

Sequencing

3. A sequencing cleanup method, e.g., Ethanol–EDTA (per the BigDye manual ), sephadex-based methods such as Qiagen DyeEx.

4. Refrigerated plate centrifuge (depends on cleanup method selected).

2.5. Cloning

1. TOPO-TA cloning kit (Invitrogen).

(Using the TOPO-TA

2. Agar plates containing X-gal, IPTG, and Ampicillin per the
Kit, Invitrogen)

TOPO manual for blue–white screening of plasmid-containing colonies.

3. PCR r
eagents as listed in Subheading 2.1 available in the mod-

ern lab (hot-start
Taq
and BSA are not necessary).

4. Water bath.

5. Incubator.

6. Bunsen burner.

7. Fumehood (recommended for handling bacteria, but not

required).

3. Methods

While setting up the reactions, open the reagent containers, tip boxes, PCR tubes, etc.,
only
when pipetting in or out. This will greatly reduce any potential contamination transmitted by aerosols.

3.1. Master Mix Setup

1. As all of the PCRs will use the same basic recipe, plan out a master mix of the ingredients common to all reactions (see Note 3). Always include at least one PCR negative control (no DNA extract) reaction per 8–10 sample reactions (see Note 4).

Generally, PCR positive controls are avoided in aDNA.

However, if a positive control is necessary, use another ancient sample as this control (see Note 5).

(a)

1–2 units of polymerase (<0.25 μ L Platinum Taq High

Fidelity or AmpliTaq Gold) (see Note 6).

(b) 1× buffer (2.5 μ L 10× PCR buffer).

(c)

0.25–0.625 mM each dNTP (0.25–0.625 μ L of 25 mM

dNTPs).

(d) 2–4 mM magnesium (i.e., 1–2 μ L of MgSO for HiFi or 4

2–4 μ L of MgCl for AmpliTaq Gold) (see Note 7).

2

15 PCR

Amplifi cation, Cloning, and Sequencing of Ancient DNA

115

(e)

1–2 μ g serum albumin (2.5–5 μ L of 10 mg/mL solution).

(f)

Purifi ed water to 25 μ L; accounting for primers and template volume (below).

Follow the manufacturer’s protocol provided with the

polymerase to set up a 25 μ L reaction. This will generally involve the following:

2. Mix the master mix by fl icking it with a fi ngertip and inverting the tube several times. Dispense the master mix into all of the tubes.

3. If only one set of primer is used, add them to the reaction mix: 0.2–0.4 μ M each primer (0.5–1 μ L of 10 μ M stock). If not, add the primers to each set of reactions separately by preparing a primer mix in the cap of the PCR tube of the negative and then dispense into the caps of the reactions that are designated to contain sample.

4. Add template to tubes individually. Ensure all tubes are closed before opening any of the template tubes (see Note 8).

Dispense the template directly into the mix in the tube.

Generally 0.5–1.0 μ L (1–5% of the DNA extract’s total volume) of template is used (see Note 9).

5. Spin down the reaction tubes briefl y, and place them in the thermocycler. Use the basic cycling conditions as suggested for the polymerase manufacturer, paying attention to include an initial hot-start period, if applicable (see Note 10). Due to the generally low number of initial template molecules, increasing the number of cycles may provide greater yield. It is common to perform 40–60 PCR cycles with aDNA.

3.2. Agarose Gel

1. Prepare a 2% or higher concentration agarose gel in TAE (or
Visualization and PCR

TBE). EtBr may either be included in the buffer, the gel, or
Purifi cation

applied as a post-stain. EtBr is a mutagen and must be handled with care (see Note 11).

2. Run out 2–5 μ L of the completed PCR (see Note 12). Visualize with UV light. If bands of the expected length are present in the negative(s), repeat the PCR. If no bands are present at all, try increasing or decreasing either the stringency of the reaction or the amount of template used (see Note 9). If many bands are observed, an increase in stringency is required (use less magnesium and/or increase the annealing temperature).

3. If the PCR yields a single, clean band, purify the remaining 20 μ L of the reaction by removing any unincorporated reagents via your favorite commercial PCR purifi cation system. To

obtain a more concentrated PCR product for direct sequencing, elute the reactions in less eluate than normal (~50% of normal eluate volume).

116

T.L. Fulton and M. Stiller

3.3. Direct Sequencing

1. Quantify the product using a nanospec or by visual estimation on agarose (compared to the ladder of known concentration).

2. Sequence using BigDye v3.1 (or the latest version) chemistry and recommended cycling conditions. This chemistry can be

diluted for economy, because long reads are not necessary for the short aDNA PCR target fragments. Due to small volumes, this should be prepared as a large master mix based on a single reaction:

(a)

0.25 μ L BigDye v3.1.

(b) 1.75 μ L BigDye v3.1buffer.

(c)

6.4 μ L purifi ed template + water (~30–60 ng template

works well).

(d) 1.6 μ L 1 μ M primer.

3. Purify the sequencing reaction using EDTA–Ethanol precipitation as described in the
BigDye manual
or by sephadex-based methods. Depending on the strength of the reaction, 50–100%

of the sequencing reaction will need to be loaded for detection on a capillary sequencer (see Note 13).

3.4. Cloning

1. Clone PCR products using any PCR product cloning protocol.

TOPO-TA cloning (Invitrogen) is quick, easy, and effi cient, but expensive (see Note 14).

2. Plate out up to three plates from each cloning reaction, depending on the dilution factor of the TOPO kit. If the reaction is scaled down tenfold, plating the entire reaction on a single plate generally yields an appropriate number of colonies.

3. Fill a 96-well plate with 50 μ L of water in each well. Any colonies that have grown up in the presence of ampicillin will contain the plasmid, which confers ampicillin resistance. By invoking a traditional blue–white screening protocol based on the modifi ed
lac
operon system, colonies with plasmids lacking a PCR insert will be blue (all β -galactosidase enzyme fragments are produced and the substrate, X-gal, is cleaved and turns color) and colonies that hold the PCR insert will be white (no lacZ α fragment is produced and X-gal is not cleaved). Pick 7–15 positive (white) colonies with separate pipette tips, placing each tip into the water of a separate well (see Note 15).

4. Place a micropipette onto the tip, swirl the tip in the water, and then blow out any liquid in the tip to ensure that the colony has transferred to the water. Eject the tip into biohazardous waste receptacle. Pick one negative (blue) colony per cloning reaction for easy visual comparison on the gel with the colonies that should have the short aDNA insert. Cover the plate with a sticker or lids and vortex vigorously to break up the colony and resuspend the cells. There is no need to heat the cells as they will lyse upon initiation of PCR cycling.

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Amplifi cation, Cloning, and Sequencing of Ancient DNA

117

5. Amplify the insert using appropriate primers for the vector used (M13 for TOPO-TA) in the 12.5 μ L reaction: 1× buffer, 0.125

μ L regular
Taq
(i.e., Promega, Applied Biosystems, EconoTaq), 0.125 μ L 25 mM dNTPs, 0.5 μ L 25 mM MgCl ,

2

1.25 μ L 10 μ M each primer, 5 μ L of colony solution, and water up to 12.5 μ L total volume.

6. Visualize and clean the PCR product as for the fi rst PCR, but only load 2–3 μ L of PCR product on the gel.

7. Sequence as above, but only use 0.5–1 μ L of template and only load 30–50% or less of the reaction for capillary sequencing.

4. Notes

1. It is easier to optimize reactions when the magnesium is not included in the buffer.

2. Although BSA is not required for PCR to function, it is almost always used in aDNA PCR.

3. As polymerase is very expensive, there is no need to make extra mix unless you are doing many reactions at a time. If signifi -

cant losses due to pipetting occur, add an extra reaction to the mix recipe.

4. Carrier DNA, such as lambda phage DNA or DNA from

another nontarget species, can be added to the PCR negative to ensure that any contaminants that may be present are

amplifi ed.

5. If there is no choice but to use a modern sample as a PCR positive, set up all the reactions, take the tubes to the modern lab and open
only
the single tube to which the template is to be added. Be sure to include a modern positive control that will be easily identifi ed as a contaminant (i.e., a genetically divergent sample). Modern samples should
never
be brought into the ancient lab.

6. Do not vortex or centrifuge the polymerase itself, only as part of the master mix.

7. If you are not using a hot-start
Taq
, the magnesium can be added separately with the primers in the reaction tube cap and only spun down immediately before cycling. Alternately, a small amount of sterile wax can be added to the master mix and

melted, briefl y, and the primer/
Taq
mix added on top of this protective barrier. Either of these will help to reduce the chance of nonspecifi c polymerase activity. A non hot-start
Taq
polymerase reaction should be set up on ice or in a cold block.

8. Tubes with individual caps are useful for this.