Physical Anthropology 167 – 172
Ethanol re-precipitation removes PCR
inhibitors from Ancient DNA extract
Rajeev Kumar Pandey
12, Deepankar Pratap Singh
35,
Godi Sudhakar
2, Vadlamudi Raghavendra Rao
14*Abstract
One of the major problems in ancient DNA work is the presence of inhibitory substances, which hampers Taq polymerase activity. Therefore analysis of ancient DNA sample is very challenging. Here we describe a simple and competent ethanol re-precipitation based protocol for the purification of DNA from ancient bones and tissues. The efficiency of this procedure has been demonstrated on 600 years old biological samples provided by Anthropological Survey of India (Himalaya region). This suggests that re-precipitation of ancient DNA extracts removes PCR inhibitors and increases the success rate of amplification.
Key words: PCR inhibitors, Ancient DNA, Re-precipitation, PCR
INTRODUCTION
Analysis of contemporary organisms can provide indirect evidence of the evolutionary history, and the reconstructions remain tentative if they cannot be checked against fossil records. Although working with ancient samples could unravel the molecular evolution, there are several technical limitations that make the study extremely difficult. Very small amount of DNA, the decay of molecules over time, post-mortem modifications and contamination with DNA from other organisms has proven to be considerable hurdles for these studies (Hansen et al. 2006; Zhang and Wu 2005; Paabo 19:9; Paabo. et al. 2004; Binladen et al. 2005; Hanni et al. 1995; Tbor Kalmar et al. 2000; Willerslev and Cooper 2005; Hebsgaard et al.2005; Kaestle. and Horsburgh. 2002). A major factor, which affects PCR amplification, is the presence of inhibitors, which vary between state of preservation. These inhibitors are result of contaminants from the surrounding environment of samples in the form of humic acid,
1 Anthropological survey of India, Mysore, India
2 Department of Human Genetics, Andhra University, Visakhapatnam 530003, India
3 S2 Functionnel Genomics,IBENS, UMR:197,Ecole Normale Superieure 46, rue d'Ulm, 75005 Paris, France 4 Department of Anthropology, University of Delhi, Delhi – 110007, India
5 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007,India
fulvic acid, hydroxi-apatite, tannin and contaminating DNA, or from degradation in the biological sample itself. This could also be due to influence of terrestrial heat (Hansen et al. 2006; Zhang and Wu 2005; Paabo et al. 2004; Shinoda et al.2006). Various oxidative processes, which modify the ancient DNA, are responsible for the low recovery rate of undamaged DNA from the archeological remains. Collagen type-I and Maillard products are the inhibitory factors of PCR amplification in case of bone (Paabo 19:9; Paabo et al. 2004; Willerslev and Cooper 2005; Kemp . and Smith et al.2005; Kaestle. and Horsburgh 2002).
In most of the published protocols almost the extraction procedures are same (Binladen et al. 2005; Hanni et al. 1995; Ovchinnikov and Goodwin et al. 2001; Cappellini et al.2004). After extraction the aqueous phase was concentrated with micro concentrators, precipitated by silica suspension/glass milk (Gene Clean Spin Wash) (Shinoda et al.2006; Thomas et al. 2004). Some commercial kits are also available which are dedicated for the extraction of ancient DNA such as Gene Clean Spin Kit for Ancient DNA (BIO101). Another study shows that sodium-acetate-isopropanol extraction was possibly better than phenol-chloroform method which gives three times more yield than glass milk method (Hanni et al. 1995). One more protocol was published, in which Dextran blue is used, for removing PCR inhibitors with selective ethanol precipitation (Kalmar et al. 2000).
Despite the availability of several methods for the analysis of ancient DNA samples, still the problems of extraction and amplification remains obscure. The efficiency of every protocol has its own limitations, depending upon the condition of the experimental specimen. We have observed that the main problem dealing with ancient DNA is the unsuccessful/irreproducible amplification. Sometimes we succeed to amplify some samples; however, these samples are not reproducible after some hours. This may be due to generation of some sort of inhibitors in the DNA solution over the time. Even if one gets the fair amount of DNA, no one can predict that it can be amplified owing to accumulation of several PCR inhibitors. Every extract coming from these precious specimens is worthy and we cannot thrust them aside.
Therefore, we have been trying to develop a simple protocol to overcome the PCR problem and successfully developed a simple and efficient re-precipitation method for ancient DNA amplification. A very simple and efficient re-precipitation method is described in this study for the purification of DNA from ancient bone samples. In our adjudication this protocol is better in so many perspectives like; re-use of the extracts which were not giving amplification but DNA is there, loss of DNA in the process is very less, no involvement of hazardous organic solvents, special devices, or enzymes which increases the possibility of contamination and DNA degradation.
MATERIALS AND METHODS Ancient specimens
Ancient Human bone samples were kindly provided by Anthropological Survey of India and all experiment was performed in ancient DNA laboratory of the Centre for Cellular and Molecular Biology (CCMB), Hyderabad. These samples are derived from Himalayan region of India and was preserved at 4˚C for further work
Contamination Precautions
Standard contamination precautions strictly followed in ancient DNA studies were performed during the experiments (Paabo et al. 2004; Kalmar et al. 2000; Willerslev and Cooper 2005; Kemp. and Smith et al.2005; Kaestle and Horsburgh 2002; Cappellini et al.2004; Shinoda et al.2006; Thomas et al.2004; Kemp and Smith 2005; Willerslev , Hansen . and Poinar 2004).
DNA Extraction
fragments were mechanically pulverized into a fine meal in sterile pestle-mortar. The samples (500mg) were then soaked in 5ml of 0.5M EDTA (pH :.0) with 200µl of 20mg/ml Proteinase K and 120µl of 10% SLS (Sodium Lauryl Sulphate) at 55˚C for overnight.
Two different methods were employed for the extraction of DNA from the powdered samples. In the first method decalcified samples were lysed in 2ml of Lysis buffer (10mM tris HCl ph :.0, 1mM NaCl) at 60˚C for 24 hrs, which later subjected to followed by phenol, phenol-chloroform, chloroform-isoamyl alcohol extraction. At last, the aqueous phase was concentrated by centrifugation driven dialysis using Centricon-30 micro concentrators from Amicon. The concentrate, which was about 0.7ml, purified by Gene Clean Spin Kit and later eluted in 0.1m Tris-EDTA. In the second method extraction was done with Gene Clean Kit for Ancient DNA (BIO 101). The pellet of decalcified bone samples were used for DNA extraction with kit. Approximately 40µl of DNA extract was obtained and visualized on 1% agarose gel (Figure 1).
Fig. 1
Agarose DNA electrophoresis (1%): [Lane 1: λ phage DNA marker, lane 2: DNA sample extracted by Gene Clean Kit, lane 3: Blank, lane
4: DNA sample extracted by phenol- Chloroform method].
Re-precipitation of DNA extracts
As we found that the DNA samples extracted using the above methods failed with PCR amplification, we have introduced a re-precipitation protocol for purification. For the handiness, volume of extract was made up to 200µl and equal volume of chloroform-iso-amyl alcohol was added to extract. The aqueous phase, after centrifugation at 12,000 rpm for 15 min, was transferred to 1.5ml eppendorf ’s tube and 1/23rd volume of 3M-sodium acetate, 2 volume of ethanol was added and kept at -20˚C for
overnight. To pellet, solution was centrifuged at 14,000 rpm for 15 min. supernatant discarded and pellet washed with :5% ethanol and spin again at 14,000 rpm for 10 min. Supernatant discarded and allowed to dry and dissolved in 40-50 µl of 0.1M Tris-EDTA.
PCR and Sequencing
The PCR amplification was carried out in 20μl reaction volume containing 1μL of re-precipitated template, 100μM each of dNTPs, 4 pM of each primer, 1X PCR buffer [100mM Tris-HCL, pH :.3 (at 250C), 500mM KCl, 15 mM MgCl
2, 0.01%(w/v) gelatin, bovine serum albumin (BSA) (10 mg/ml)
(New England Bio Labs) and 0.5 units of AmpliTaq Gold (Applied Biosystems). Bovine serum albumin was used as it can bind to various types of PCR inhibitors that might have carried during extraction. Amplification of Hypervariable Region (HV region) of bone samples was carried out using the primers listed (Table 1).
RESULTS AND DISCUSSION
Since the commencement of ancient DNA work several methods have been demonstrated to make them easily extractable, amplifiable and inhibitors free (Hanni et al. 1995; Kalmar et al. 2000). However, still there is no such protocol, which fulfills all these demands in one. One, which passes one hurdle, fails in second. Hanni et al. 1995, published an isopropanol-based precipitation method (Hanni et al. 1995) that eliminates time-consuming dialysis and microconcentrator step. Kalmar et al. 2000 demonstrated that precipitation of sample with Dextran blue removes PCR inhibitors as well as save the time of decalcification step (Kalmar et al. 2000). Hanni et al. 1995, noticed the presence of blurring blue florescence (derived from Maillard products of reducing sugars), which migrates at the level of 500bp DNA molecule, and they correlated them with PCR inhibitors activity. In our case we didn’t get such type of florescence at 500bp (Figure 1) level in our samples, but still they were showing inhibitory activity.
We have demonstrated that 1µl of ancient DNA extract can inhibit the PCR reaction using modern DNA as a template. If the ancient DNA extract is progressively diluted (1/10, 1/7, 1/5, 1/2) and added to the reaction mixture containing contemporary DNA AmpliTaq Gold polymerase activity was gradually restored. It means that inhibitors are present in fair amount and with gradual dilution inhibition was decreased. The same sample was introduced for amplification with mitochondrial DNA primers (Table n. 1)
Table 1
Primer Name Primer Sequence
F15996 5’CTCCACCATTAGCACCCAAAGC 3’
R16410 5’GCGGGATATTGATTTCACGG 3’
Sequences of the primer designed for the ancient DNA study
Which was purified by the present method and good yield of PCR product (5µl were loaded on 2% agarose gel) was clearly seen that the re-precipitated extract gives amplified products of the expected size after amplification (Figure 2). So, the method works for the samples, which were in much battered condition and proves to be very effective method for ancient DNA.
Fig. 2
There was no need of two consecutive PCR amplification i.e. re-amplification, as we were getting good yield in first amplification (40 cycles). The original extract has not given any amplification but the re-extracted samples has given proper amplification and interestingly we succeeded to amplify a long stretch of 424 base pairs which is not usual case with ancient DNA samples (Kalmar et al. 2000). This could be because of proper purification of DNA, which removed prominent PCR inhibitors efficiently. This will reduce the time and labour in PCR and sequencing of two overlapping sets into a single amplicon stretch of same size. All the amplified PCR products of varied lengths were successfully sequenced and assembled with Cambridge reference sequence (r-CRS) using autoassembler (ABI), which shows mitochondrial identity (Figure 3).
Fig. 3
A
B
Assembly of ancient Sample with r-CRS: Two Polymorphism were found in our ancient sample (A) g.16147: C>T; (B) g.16223: C>T
Acknowledgment
The authors thank Dr K.Thangaraj, Scientist, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India for providing all the lab facilities. The financial assistance of Anthropological survey of India is gratefully acknowledged.
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