Download Isolation and amplification of ancient DNA

Isolation and amplification of ancient DNA
Methods of ancient DNA isolation
Ancient DNA (aDNA) analyses rely on the extraction of the minute amount of DNA remaining in a sample which can be hundreds to tens of thousands of years old. Obviously, the
efficiency of DNA extraction from archaeological sample plays a key role for the whole analytical process. aDNA studies share a common feature with forensics and other approaches
basing on analyses of museum and non-invasively collected specimens – the amount of endogenous DNA available in the samples is often limited. Thus, techniques that provide extraction of the highest possible amount of DNA molecules from a specimen are of crucial importance. Consequently, most of extraction methods use a buffer containing EDTA for bone
decalcification and proteinase K for protein digestion. After such a common step obtained
preparation can be treated by a wide range of methods which are currently in use including
and relying on various techniques such as filtration on spin columns, alcohol precipitation,
binding to silica, phenol-chloroform extraction, binding to magnetic beads. The latter method
was applied in construction of semi-automatic biorobots isolating DNA. Isolation methods
used by various teams working with aDNA vary between each other; however, the silica
method is the most popular one and is most commonly used in different variants. Basing on
many reports, one can conclude that selection of isolation method depends mainly on the
sample characteristics and its origin is critical for the choice.
In order to prevent possible contamination, which source are people involved in the
process, all stages of the work must be carried out under sterile conditions which implies the
use of disposable gloves and mouth masks.
Phenol-chloroform method
The method described in this protocol, i.e. the phenol-chloroform method, is based on denaturing properties of phenol and chloroform. After mixing and centrifugation denatured proteins are found in organic phase or interphase, while DNA remains in the aqueous phase. Isoamyl alcohol improves the separation of phases and prevents foaming.
Precipitation method
Precipitation of DNA with salt and ethanol is a commonly used method for concentrating
nucleic acids. Salt and ethanol are added to aqueous solution to precipitate nucleic acids from
solution. Solution is centrifuged and precipitate of nucleic acids is than washed with cold 70%
ethanol to remove the salt.
Water (H2O) is a polar molecule that has a partial negative charge near the oxygen atom due to the
presence of unshared pairs of electrons, and partial positive charges near the hydrogen atoms. Due to
this polar nature of water, other polar molecules such as DNA or RNA can interact electrostatically
with the water molecules, allowing for easy self-dissolving in water. Precipitation breaks existing interaction and as a result forms DNA aggregates easily isolated after centrifugation as a pellet.
(SALTS) The role of salts in the mentioned method is to neutralize charges of sugar-phosphate
backbone of the nucleic acid. Sodium acetate is a commonly used salt for DNA and RNA precipitation. In aqueous solution, sodium acetate hydrolyses into Na+ and CH3COO-. Positively charged sodium ions neutralize negatively charged backbone of DNA, in particular PO3- groups of nucleic acids,
changing them into far less hydrophilic molecules, and therefore much less soluble in water.
(ETHANOL) Electrostatic interaction between Na+ ions from solution and the PO3- groups of nucleic acid is affected by the dielectric constant of the solution according to Coulomb’s Law. Water has
a high dielectric constant, which makes it fairly difficult for the Na+ and PO3- to interact. Ethanol on
the other hand has a much lower dielectric constant, making it much easier for Na+ to interact with the
PO3- as a result of shielding its charge. This makes the nucleic acid less hydrophilic and causes it to
drop out of solution.
Hypervariable region of mitochondial DNA – HVR
Circular mitochondrial genome, occurring in 2 to 10 copies per organelle, consists of ca 16
500 bp. The analysis of mtDNA primary structure is often used in the studies of mammal evolution due to the following features of the molecule: it is directly transmitted between generations (matrilineal lineage) and is not subject to recombination. Thus, it is possible to track the
history of single mutations. Also, due to the lack of mtDNA repair and changes introduced by
the mitochondrial polymerase, this genome is highly variable. HVR I sequence (402 bp), located in the control region between positions 15998 and 16400, is of special importance for
phylogenetic analysis. It evolves 5 to 20 times faster than the coding part of the molecule. The
place of point mutation within the HVR I defines mtDNA haplogroup the individual carrying
it belongs to, and indicates its descent.
Designed primers should flank fragments as short as it is possible (max. 200 bp), because
aDNA is highly fragmented. Low initial number of original DNA copies isolated from archaeological material causes the number of PCR cycles to increase from 30, commonly used
for amplification of modern DNA templates, to 40–50 for aDNA.
HVR I of the mtDNA control region will be amplified as two overlapping fragments:
EX I
Forward primer 16112 (5’-CGTACATTACTGCCAGCC-3’)
Reverse primer 16262 (5’-TGGTATCCTAGTGGGTGAG-3’);
EX II
Forward primer 16251 (5’-CACACATCAAC TGCAACTCC-3’)
Reverse primer 16380 (5’-TCAAGGGACCCCTATCTGAG-3’).
Obtained PCR products are 186 and 171 bp in length, respectively.
Literatura:
Sigurgardottir, S., et al. (2000). "The mutation rate in the human mtDNA control region." Am J Hum Genet 66(5): 1599-609.
Igman M., Kaessmann, H., Paabo, S., and U. Gyllensten (2000) “Mitochondrial genome variation and the origin of modern humans”, Nature, 408: 708-713
Mullis, Kary (1990). "The unusual origin of the polymerase chain reaction". Scientific American 262 (4): 56–61, 64–65
Rohland N. & Hofreiter M. (2007) “Comparison and optimization of ancient DNA extraction” BioTechniques. 42:343-352.
Kalmar T et al., (2000) ''A Simple and Efficient Method for PCR Amplifiable DNA Extraction from Ancient Bones,'' Nucleic Acids Research 28: 67-69
Cattaneo, C. et al (1995) ”A simple method for extracting DNA from old skeletal material” Forensic Sci Int 74:167–74
MATERIALS – REAGENTS – EQUIPMENT
isopropanol
PCR reagents (Taq polymerase, primers, dNTP, water)
phenol : chloroform : isoamyl alcohol (25:24:1)
sodium acetate – saturated
sodium acetate 3M, pH = 5.0
ethanol 100 % and 70%
thermal cycler
thermoblock
small centrifuge
vortex
1,5 ml tubes
gloves and mouth masks
PROCEDURE
Extraction control which is treated identically as experimental samples should be
carried out during DNA extraction process to monitor contamination with exogenous
molecules.
DNA isolation (method 1)
1. Add 250µl of saturated sodium acetate to solution of bone extract (bone powder,
EDTA, proteinase K) and vortex for 30 s.
2. Then centrifuge at 4000 x g for 10 min.
3. Transfer the supernatant containing DNA to a new polypropylene tube, add 700µl of
100% isopropanol and mix for at least 10 min to precipitate the DNA.
4. Then centrifuge at 4000 x g for 10 min.
5. Discard gently the supernatant; the remaining whitish-yellow pellet of DNA suspend
in 150 µl of 70% ethanol and place in -20 °C for 25min.
6. Then centrifuge at 13 000 x g for 10 min. at 4°C.
7. Next, dry the pallet in thermoblock at 37 °C and dissolve in 30 µl of sterile distilled
water.
8. Use the obtained solution for PCR.
DNA isolation (method 2)
1. Centrifuge the sample with bone powder solution (bone powder, EDTA, proteinase K)
at 5,000×g for 5 min
2. Transfer supernatants to a new 1,5 ml tube and add 1 volume of phenol : chloroform :
isoamyl alcohol reagent. (Be very careful with this reagent – phenol is a strong neurotoxin.)
3. Vortex mixture thoroughly.
4. Centrifuge the solution at 10 000 x g for 2 min.
5. Carefully transfer aqueous phase (top) to a new tube
6. Repeat steps (2-5) until there is no precipitate in interphase.
7. Next, concentrate aqueous phase precipitating by the addition of 0.1 volume of
3 M sodium acetate at pH 5.0 and 2.5 volume of cold ethanol.
8. After mixing, incubate the sample at -20ºC for 25 min. and centrifuge at 15,000 x g for
10 min at 4°C.
9. Discard gently the supernatant; the remaining pellet of DNA suspend in 150 µl of 70%
ethanol and place in -20 °C for 15min.
10. Then centrifuge at 13 000 x g for 10 min. at 4°C.
11. Next, dry the pallet in thermoblock at 37 °C and dissolve in 30 µl of sterile distilled
water
12. Use the obtained solution for PCR.
DNA amplification (PCR)
1. Mix the following components for each reaction in a 0.2 ml tube (add polymerase as
the last one):
Reagent
Volume
Concentration
H2O
15,7 µl
-
Buffer
2,5 µl
10x
dNTP
2 µl
2,5 mM
Primers
0,3 µl each
10 µM
DNA extract
4 µl
-
Taq Polymerase
0,2 µl
5 U/µl
2. Prepare a control reaction with 4 μl of sterile water instead of sample (DNA).
3. Place tubes in a thermal cycler preheated to 94oC and start applying proper times and
temperature as stated below:
HVR I
Number of cycles
38
Initial denaturation
94ºC 4min
Denaturation
94ºC 40 sec
Anneling
55ºC 30 sec
Extention
72ºC 45 sec
Final Extention
72ºC 7 min
Hold
4ºC ∞