Ancient DNA: Methods and Protocols (26 page)

Recently, the method was coupled with a new method of generating barcoded sequence libraries for sequencing on next generation high-throughput sequencing platforms (i.e., Roche’s 454 or Illumina’s Solexa; see Chapter 20
( 16 )
).

The fi rst step of the two-step amplifi cation strategy is the multiplex PCR. In this step, every second fragment of a series of overlapping fragments is amplifi ed by adding the appropriate primer pairs into one of two separate, nonoverlapping mixtures, commonly referred as to the “odd” and the “even” set. Importantly, overlapping fragments must not be amplifi ed in the same PCR, because the forward primer of fragment 2 can pair up with the reverse primer of the upstream fragment 1. The odd set therefore includes the primer pairs for fragments 1, 3, 5, and so on, and the even set includes the primers amplifying fragments 2, 4, 6, and so on. Thus, only two PCRs need to be performed, compared to a single reaction for each primer pair in standard PCR.

A dilution of this fi rst-step multiplex amplifi cation reaction serves then as template for the subsequent second-step amplifi cations. In the second step, each of these individual PCRs (also called simplex, singleplex or monoplex PCRs) includes only a single primer pair to be amplifi ed. Because the starting template is the amplifi ed product from the fi rst-step reaction, rather than the original DNA extract, the amount of the original DNA extract that was necessary to amplify all of the targeted region (or regions) is dramatically reduced.

17 Multiplex PCR Amplifi cation of Ancient DNA

135

2. Materials

2.1. PCR Reagents

All reagents and plastic consumables must be sterile (DNA and DNAse free) and of molecular biology grade or similar.

1. Deoxynucleoside triphosphates (dNTPs) of 100

m M each,

combined in equal volume to yield a dNTP mix of 25 m M

each dATP, dGTP, dTTP, and dCTP.

2. DNA polymerase + buffer supplied with polymerase, usually AmpliTaq Gold (see Note 1).

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

2

4

4. Forward and reverse primers in 100 m M stock solutions (see Note 2) as well as diluted to 10 m M.

5. Serum albumin (SA), e.g., bovine (BSA) or rabbit serum albumin (RSA), prepared as 10 mg/mL solution in sterile water (see Note 3).

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

7. Thermocycler with heated lid.

8. DNA template.

9. Flowhood or PCR-free region in the modern laboratory for the second-step PCR setup.

2.2. Agarose Gel

1. 2% Agarose gel.

Visualization and PCR

2. 1× TAE (Tris–acetate–EDTA) or 0.5× TBE (Tris–borate–

Purifi cation

EDTA) running buffer, available commercially.

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 m l (0.25 m g) prepared ladder + 1125 m L TE + 250 m L 6× loading dye (supplied with ladder).

5. Agarose gel electrophoresis rig and power supply.

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

7. Your preferred commercial PCR purifi cation kit: Qiagen, Millipore, ExoSAP, Agencourt AMPure XP.

3. Methods

3.1. Primer Design

1. Design a series of overlapping primer sets that do not vary by more than 3°C in their melting temperatures, as they will all be cycled at the same temperature. The targeted regions should 136

M. Stiller and T.L. Fulton

generally range in length between 100 and 300 bp excluding primers. Divide the primer sets into the “odd” and “even” sets.

These sets can be used for both the fi rst and second-step PCRs (see Note 4).

2. To maximize specifi city and selectivity for the desired target fragments, a second set of primers can be designed for use in the second-step PCRs (instead of using the same set of primers in both steps). In this case, one or both primers used in the second-step amplifi cations would be partially or even fully nested within the target fragment (see Note 5).

3.2. First-Step PCR

1. In an aDNA clean room, prepare the primer sets by mixing all
Setup

the forward and reverse primer pairs of the “odd” set together to obtain a working primer solution in which each primer will have a fi nal concentration of 1 m M in the primer solution (see Note 2). Do the same for the “even” set.

2. Prepare the aDNA multiplex PCR master mix (add all reagents except the DNA template) as outlined in T
able 1
. Prepare suffi cient master mix for all samples plus a PCR negative control reaction for every 8–10 sample reactions (see Note 6).

3. Dispense the master mix into the PCR strip tubes or plates, and then close all the lids.

4. Add DNA template individually to each reaction tube, opening only one tube (or row of tubes, if using multichannel pipettes) at a time to avoid cross-contamination.

5. Before leaving the clean room, set up the second-step PCR

r
eaction mix as described in Subheading 3.3
. The fi rst-step Table 1

First-step multiplex PCR setup

Reagent

Volume ( m L) per sample

Final concentration in reaction

Water (add to make 20 m L fi nal

reaction volume)

10× buffer

2

1×

25 mM MgCl

2–3.2

2.5–4 mM

2

BSA or RSA (10 mg/mL)

2

1 mg/mL

dNTPs (25 mM each)

0.2

0.25 mM each

Primer mix (1 m M each primer)

3.0

0.15 m M each primer

AmpliTaq Gold (5 U/ m L)

0.4

2 U

Template

1–5

17 Multiplex PCR Amplifi cation of Ancient DNA

137

reactions can be kept in cold while you prepare these reactions (see Note 7).

6. After the fi rst-step PCR is completely set up and the second-step PCR is missing only the template DNA (this will be the result of the fi rst-step PCR), proceed to the modern lab. Cycle the fi rst-step reactions using an initial step of 95°C for 12 min to activate the hot-start polymerase, followed by 30–40 cycles of denaturation for 30 s at 94°C, primer annealing for 30 s at the required annealing temperature determined by your primer set, and elongation for 40 s at 72°C. Finish the cycling protocol with a fi nal extension step at 72°C for 4 min (see Note 8).

7. After the cycling is fi nished, dilute a part of the fi rst-step reactions 1:20–1:50 (depending on how many second-step reactions you are planning to set up) with clean water (see Notes 9

and 10). This will be the template for the second-step PCR.

3.3. Second-Step PCR

1. Plan the plate layout. If suffi cient samples are being processed
Setup

that multichannel pipettes will be used, set up the second-step PCR so that the fi rst-step reactions can be diluted and dispensed straightforwardly using the multichannel pipette (see Note 11).

2. In the clean room, set up the second-step reaction master mixes (also called monoplex, simplex, or singleplex PCRs) for each single primer pair separately following the recipe outlined in T
able 2
. Include suffi cient mix for at least one PCR negative control per 8–10 fi rst-step reactions to monitor any contamination introduced during the process of diluting the fi rst-step Table 2

Second-step multiplex PCR setup

Reagent

Volume ( m L) per sample

Final concentration in reaction

Water (add to 20 m L)

10× buffer

2

1×

25 mM MgCl

2–3.2

2.5–4 mM

2

BSA or RSA (10 mg/mL)

2

1 mg/mL

dNTPs (25 mM each)

0.2

0.25 mM each

Forward primer (10 m M)

1.5

0.75 m M

Reverse primer (10 m M)

1.5

0.75 m M

AmpliTaq Gold (5 U/ m L)

0.1

0.5 U

Template (dilution of fi rst-step PCR)

5

138

M. Stiller and T.L. Fulton

PCR or when adding the dilutions as a template to the second-step reactions. No template is added yet.

3. Dispense the master mix and seal the tubes or plates. Take this master mix to the modern lab (see Note 12).

4. After the fi rst-step PCR is complete and the reactions are
diluted (Subheading 3.2
, step 7), add the template to the second-step reactions in a laminar fl ow hood or area free of post-PCR processing (like agarose gel visualization) that is
not
the aDNA lab (see Note 13).

5. Perform the second-step PCR reactions using the same cycling conditions as used for the fi
rst-step PCR in Subheading 3.2 ,

step 6 (see Note 8).

3.4. Agarose Gel

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

TBE). Ethidium bromide (EtBr) may either be included in the
Purifi cation

buffer, the gel, or applied as a post-stain. EtBr is a mutagen and must be handled with care (see Note 14).

2. Run out 3 m L of the completed second-step PCR and the PCR

negative controls from the fi rst-step reactions (see Note 15).

Visualize with UV light. If bands of the expected length are present in the fi rst-step negative(s), repeat the fi rst-step PCR

for the particular primer set. If bands are present in the second-step negative(s), repeat the second-step PCR for the particular primer pair.

3. If the second-step PCR yields a single clean band, purify the remaining 17 m L of the reaction using your favorite commercial PCR purifi cation system (see Note 16).

4. The PCR products can now either be cloned or directly sequenced using traditional Sanger sequencing or processed with high-throughput sequencing methods, potentially incorporating a barcoding step.

4. Notes

1. It is easier to optimize reactions when the magnesium is not included in the buffer, as changing the concentration of magnesium is a useful optimization protocol.

2. If you intend to use more than 100 single primers (i.e., 50

primer pairs) in a single primer set, a more concentrated stock solution is required, since the fi nal concentration of each primer within the primer set should be 1 m M.

3. Although serum albumin (SA) is not required for PCR, it is almost always used in aDNA PCR.

17 Multiplex PCR Amplifi cation of Ancient DNA

139

4. Due to the abundance of short aDNA template molecules in the extracts, the overlaps between adjacent fragments may be preferentially amplifi ed compared to the desired long fragments and thus outcompete them in the course of the PCR

reaction. In case of a very tight tiling of very short target fragments, three or four different primer sets, rather than the “odd” and “even” sets described here, may be necessary.

5. This strategy is particularly useful when attempting to amplify nuclear loci. When targeting mitochondrial fragments, the

identical primers from the fi rst-step amplifi cations are generally used in the second-step PCRs.

6. As in standard PCR, it may be benefi cial to add carrier DNA (e.g., lambda phage DNA or DNA from another nontarget

species) in the PCR negative controls to exclude potential carrier effects that may prevent detection of contamination. PCR

positive controls should be avoided if possible.

7. If the mix for the second-step PCR is to be prepared on a different day, the fi rst-step PCR can be stored in the freezer in the modern lab.

8. The cycling conditions provided are specifi c to AmpliTaq Gold (Applied Biosystems). If a different polymerase is used, adjust the cycling conditions according to the manufacturer’s recommended activation and extension temperatures.

9. The water used to dilute the fi rst-step reactions should be sterile. We recommend bringing water in strip tubes or in plates directly from the clean room, where it can be prepared while setting up the PCR. This avoids carryover contamination in the second-step reactions due to the use of potentially contaminated water from the post-PCR facilities.

10. Performing two second-step reactions from the same fi rst-step template is not a replication of the PCR, since both second steps start from the same initial template molecule(s) amplifi ed during the fi rst-step reaction. Two independent, fi rst-step amplifi cation reactions must be performed to meet the criteria of independent replication.

11. For example, if eight samples are processed in the fi rst PCR, set up the second PCR so that sample 1 is the template for 12

singleplex reactions in row A of a 96-well plate, sample 2 is in row B,
etc.
Thus, singleplex primer set 1 will be used for column 1 on the plate, primer set 2 in column 2,
etc.

12. Using a hot-start polymerase like AmpliTaq Gold (Applied Biosystems) makes it possible to set up the second-step reactions in the clean room immediately after the fi rst-step reactions are prepared. The second-step reaction mix can then be stored with sealed lids outside the clean room until the fi rst-step reactions are fi nished, and amplifi ed products from the 140

M. Stiller and T.L. Fulton

fi rst step can be added to the prepared second-step reactions at that point.

13. As the template for the second-step PCR is already a PCR-amplifi ed product, it is critical that it is not brought back into the clean room, which must stay free of PCR product. However, the reactions are still highly sensitive to contamination and must be prepared with caution, in the same manner that one would behave in the clean room (i.e., opening tubes only when necessary).