Ancient DNA: Methods and Protocols (23 page)

118

T.L. Fulton and M. Stiller

9. As aDNA extractions frequently contain many inhibitors, often reducing the amount of template can increase product yield as inhibitors are more diluted. This may be achieved by either reducing the amount of template in the PCR reaction or by

diluting aliquots of the extract itself by a factor of 5–10.

10. In case of using AmpliTaq Gold, increasing the initial hot-start period from 10 min (as recommended by the manufacturer) to 12 min will give more consistent amplifi cation results, especially when the reaction is starting from a very low number of initial template molecules.

11. Adding EtBr to the running buffer offers the most dilute option, but increases the potential for splashing and also leads to EtBr contamination of the gel apparatus. Addition of EtBr to the gel prior to setting also contaminates the apparatus, but keeps the EtBr more contained than in the buffer. However, if EtBr is added to the gel, it must
never
be added before heating, as EtBr will be aerosolized, which is hazardous. Staining the gel after electrophoresis requires a more concentrated solution of EtBr than would be added to the running buffer, but provides a very contained region of contamination and often produces sharper DNA band images. Always dispose of EtBr-contaminated waste following your institution’s health and safety protocols.

12. Be sure that the samples are run out slowly so adequate separation occurs. As fragments are quite small, it is important to obtain clear differentiation from primer-dimers, which are often not much smaller than the desired product.

13. Do not load this much modern sequence—you will be greatly overloading the reaction, which is hard on the sequencer.

14. Although it is not recommended by the manufacturer, we have had success using reduced reaction volumes. The reaction can be scaled down up to tenfold, but the results become increasingly erratic with increased dilution, presumably as the reaction kinetics become less effi cient. For ten cloning reactions from one tube of competent cells, follow manufacturer’s protocol for TOPO-TA cloning, but modify the following volumes: use 3.5–5 ng of DNA in a total of 0.5 μ L, plus 1 μ L

prepared reaction mix (1.25 μ L salt, 1.25 μ L vector, 7.5 μ L

water), and 5 μ L competent cells. Recover in 50 μ L SOC or LB media and plate everything on a single agar plate.

15. If the sample is known to be uncontaminated and not badly damaged, fewer (i.e., 3–4) clones are required. If the sample is thought to have high damage or low-level contamination from collection or storage, more clones are required (i.e., 12–16).

With human work, even more clones (24+) may be desirable to detect potential contaminants.

15 PCR

Amplifi cation, Cloning, and Sequencing of Ancient DNA

119

References

1. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn

filing reveals the frequency of blocking

GT, Erlich HA, Arnheim N (1985) Enzymatic

lesions in ancient DNA. Nucleic Acids Res

amplifi cation of beta-globin genomic sequences

38:e161

and restriction site analysis for diagnosis of 5. Willerslev E, Cooper A (2005) Ancient DNA.

sickle-cell anemia. Science 230:1350–1354

Proc Biol Sci 272:3–16

2. Poinar HN, Schwarz C, Qi J, Shapiro B, 6. Poinar HN, Hofreiter M, Spaulding WG, MacPhee RDE, Buigues B, Tikhonov A, Huson

Martin PS, Stankiewicz BA, Bland H, Evershed

DH, Tomsho LP, Auch A, Rampp M, Miller

RP, Possnert G, Paabo S (1998) Molecular

W, Schuster SC (2006) Metagenomics to

coproscopy: dung and diet of the extinct

paleogenomics: large-scale sequencing of

ground sloth
Nothrotheriops shastensis
. Science

mammoth DNA. Science 311:392–394

281:402–406

3. Rohland N, Hofreiter M (2007) Comparison 7. Bartlett JMS, Stirling D (2003) PCR proto-and optimization of ancient DNA extraction.

cols, 2nd edn. Humana, Totowa, NJ

Biotechniques 42:343–352

8. Sambrook J, Russell DW (2006) The con—

4. Heyn P, Stenzel U, Briggs AW, Kircher M,

densed protocols from molecular cloning: a

Hofreiter M, Meyer M (2010) Road blocks

laboratory manual. Cold Spring Harbor

on paleogenomes-polymerase extension pro—

Laboratory Press, Cold Spring Harbor, NY

sdfsdf

Quantitative Real-Time PCR in aDNA Research

Michael Bunce , Charlotte L. Oskam , and Morten E. Allentoft Abstract

Quantitative real-time PCR (qPCR) is a technique that is widely used in the fi eld of ancient DNA (aDNA).

Quantitative PCR can be used to optimize aDNA extraction methodologies, to detect PCR inhibition, and to quantify aDNA libraries for use in high-throughput sequencing. In this chapter, we outline factors that need to be considered when developing effi cient SYBR Green qPCR assays. We describe how to setup qPCR standards of known copy number and provide some useful tips regarding interpretation of qPCR

data generated from aDNA templates.

Key words:
qPCR , Ancient DNA , SYBR Green , PCR inhibition , qPCR standard , DNA extraction optimization , Library quantitation

1. Introduction

The invention of PCR has been a crucial technological advance in the fi eld of ancient DNA (aDNA). However, conventional PCR

methods, in which the success of the reactions is evaluated at the endpoint of thermocycling (typically following 40–50 cycles), should be considered qualitative, because the dynamic range of endpoint PCR is, at best, two orders of magnitude
( 1
) . Hence, it is generally diffi cult to tell whether a PCR reaction was seeded by ten or by ten million template molecules based on the intensity of amplicon staining on a gel. Real-time PCR methods have a dynamic range of greater than eight orders of magnitude, making them a powerful analytical tool.

An in-depth discussion of the theory of quantitative PCR

(qPCR) is beyond the scope of this chapter, but can be found in a number of excellent reviews and book chapters (see
( 1– 3 )
).

In addition, we recommend the online resour
ce at http://www.

gene-quantifi cation.info/ . It should be noted that the bulk of the

Beth Shapiro and Michael Hofreiter (eds.),
Ancient DNA: Methods and Protocols
, Methods in Molecular Biology, vol. 840, DOI 10.1007/978-1-61779-516-9_16, © Springer Science+Business Media, LLC 2012

121

122

M. Bunce
et al.

a

b

Plateau phase

0.1µl extract

CT= 28.0

1µl extract

CT= 24.7

Linear phase

2µl extract

CT= 23.7

User defined

threshold

No. of amplicons (or SYBR fluorescence)

Exponential phase

0

10

20

30

40

Cycle number

CT value: 13.0

Fig. 1. (
a
) A sigmoidal PCR amplifi cation curve, depicting the exponential, linear, and plateau phases of the reaction, together with a schematic representation of the extrapolation of
C
values. The insert depicts a SYBR Green molecule (
dark T

blue
) bound to the minor groove of a DNA duplex. (
b
) A qPCR assay in which 2, 1, and 0.1 μ L (via dilution) have been placed in the reaction. The
C
values shift in accordance with the dilution factor.

T

published literature on qPCR involves research on the quantitation of gene expression via RNA, which has limited applicability to low copy number aDNA templates. This chapter will focus exclusively on the use of SYBR Green-based qPCR as opposed to the probe-based TaqMan systems, as we have found this method to be the more sensitive and cost-effective assay.

qPCR focuses on imaging the amount of amplicon present

during the exponential phase of the PCR using a fl uorescent dye (SYBR Green) that binds specifi cally in the minor groove of dou-

ble-stranded DNA (Fig. 1a ). All qPCR systems ar
e equipped with a camera that captures the fl uorescent output of the reaction at ever
y cycle. In the exponential phase (Fig. 1a ), the amplicon con-

centration, as measured by SYBR Green fl uorescence, correlates with the original level of input DNA. The output from qPCR

assays is expressed as a cycle threshold value,
C
(also called
C
), T

q

which represents the number of cycles taken to reach a certain user-defi ned threshold value in signal str
ength (Fig. 1a
). If the threshold is fi xed, the
C
values estimated from different PCRs can T

be interpreted as a relative measure of template copy numbers between DNA extracts. The threshold value needs to be defi ned at a level where the amount of fl uorescence is both detectable by the camera and is above the background level of SYBR Green binding in the r
eaction (Fig. 1b ).

16 Quantitative Real-Time PCR in aDNA Research

123

Since the
C
value images the PCR in the exponential phase, it T

should refl ect the level of input DNA (in the absence of inhibitors and assuming 100% PCR effi ciency—see below). As depicted in
Fig. 1b , if 2
μ L of an aDNA extract is aliquoted into a reaction and gives a
C
of 23.7 cycles, then adding only 1 μ L should result in a T

C
of 24.7 cycles. Likewise, adding only 0.1 μ L of the same DNA T

extract (via dilution) should yield a
C
of 28.0 cycles (a shift of 3.3

T

cycles is expected from a 1/10 dilution: 2 3.3 = 10).

The applications of qPCR in aDNA research are many, but fall into fi ve main categories:

1.
Optimization of aDNA recovery
: Using qPCR
C
values for T

absolute quantitation using either a standard or relative CT

value (see studies on bone
( 4 )
and eggshell
( 5 )
respectively), it is possible to explore different extraction protocols to maximize the recovery of DNA from ancient substrates.

2.
Detection of PCR inhibitors
: The copurifi cation of compounds that adversely affect PCR is a common problem in aDNA

research, especially in sediments (e.g., humics and tannins). By performing a serial dilution of aDNA extracts, it is possible to detect inhibition as the absence of expected
C
shifts as outlined T

above. In many instances, the apparent number of amplifi able template molecules may actually increase (resulting in a lower C
value) as the aDNA extract is diluted, as this also reduces the T

concentration of inhibitors. Extracts can also be spiked with an unrelated primer set and synthetic template to detect inhibition (internal PCR controls, or IPCs). If the IPC is positive and amplifi es at the same
C
with and without the spiked aDNA T

extract, PCR inhibition is not adversely affecting the reaction.

3.
Rapid assessment of preservation
: qPCR data can enable ranking samples in order from the best preserved to the poorest preserved by comparing
C
values. This has benefi ts in prioriT

tizing samples, identifying environments with favorable DNA preservation, and examining the effects of postmortem DNA

diagenesis in samples or stored extracts
( 6
) .

4.
aDNA authentication
: qPCR data can serve as a useful (but by no means defi nitive) means to assess data fi delity. If a reaction has amplifi ed from a very small number of starting template molecules, that reaction is likely to be more susceptible to contamination. Moreover, when dealing with very poor DNA preservation and low copy numbers of target DNA fragments, a larger fraction of the available templates may display miscoding lesions as the result of postmortem DNA damage
( 7
) .

5.
aDNA library quantifi cation
: aDNA genomic libraries constructed for high-throughput sequencing (HTS) applications (e.g., Roche 454, ABI SOLiD, and Illumina Solexa) need to

be accurately quantifi ed. Unlike modern DNA, the quantum

124

M. Bunce
et al.

of template is a limiting factor. qPCR can be used to quantify libraries both pre-and post-enrichment
( 8 )
.

In this chapter, we discuss the construction of SYBR Green qPCR assays and standards. We provide details of how to assemble the qPCR components rather than using commercially available qPCR master mixes, which offer less fl exibility in modifying reaction chemistry. In our laboratory we use qPCR to obtain information about levels of inhibition in the aDNA extract and relative preser
vation between samples

( 5, 9, 10 )
. By designing qPCR

primer pairs in phylogenetically informative regions, amplicons generated in the qPCR assay can be sequenced directly.