SAMPLE

SAMPLE COLLECTION
COLLECTION
AND
AND DNA
DNA EXTRACTION
EXTRACTION
FROM
FROM FOSSILS
FOSSILS
PRESENTED BY:
AROOJ SEHAR
ROLL No. 28
M.Sc IV
PRESENTED TO:
Dr. ISHFAQ

CONTENTS

Introduction
Sample collection
DNA extraction methods
DNA cloning
PCR
DNA extraction from fossilized bones
DNA extraction from fossils in rocks

CONTENTS

DNA extraction fossil pollen of Abies spp.

Problems for amplification of fossil DNA
Detection of inhibiting activities
Conclusion
References

INTRODUCTION

DNA extraction is the process of isolation

of DNA for scientific studies.
For
DNA
extraction
from
fossils,
compression fossils are isolated from the
rocks by using mechanical tools.
For DNA extraction, various methods are
used out of which CTAB method and
DTAB methods are mores common.

SAMPLE COLLECTION

Sample collection is the collection of

fossils from various localities, using
different techniques and methods, for
scientific study.

STEPS IN SAMPLE COLLECTION

Location of fossils
Collection Techniques
Cleaning of fossils
Documentation

1. LOCATION OF FOSSILS

Fossils are found in clastic sedimentary

rocks, non-clastic sedimentary rocks and
in evaporates.
The fossilized specimen can easily be
distinguished from rest of the rock
material because of its distinct color,
shape and texture as compared to the
rock material.

2. COLLECTION TECHNIQUES

If the rock material is stiff and hard, a

geological hammer, cold chisel and
mallet can be used.
If the fossil is deposited in soft sediments
like sand, silt or clay, spades, flat
bladed trowel and stiff brushes can
also be used.
Sieves are used for separation of fossils
from sand and gravel.

3. Cleaning of fossils

If fossils are of small size, a stiff brush is

used for cleaning, but if the specimens
are of large size then chisel can be used
for this purpose.
The use of chisel can damage the fossils,
therefore, dental tools are, sometimes
used in this process.

4. Documentation

The fossils are cataloged on the basis of

their locality number and sample number
so that they may be easily available in
catalogs of large institutions like
museums.
Data logs, photographs, and sketches of
the specimen are also helpful in
documentation.

DNA EXTRACTION

DNA extraction methods

CTAB (Hexadecyl-trimethyl-ammoniumbromide) method

DTAB (Dodecytrimethyl-ammoniumbromide)
method of Gustincich & al. (1991)
Guanidine-silica method
The DNA extraction Protocol of Doyle &
Doyle (1990)

1. CTAB (Hexadecyl-trimethyl-ammonium-bromide) method

50 mg of the tissue is ground in 50mg of

liquid nitrogen. Then eppendorf tube
containing 700pi of hot extraction buffer
is taken, the ground tissue is added and
mixed rapidly, then incubated at 600C for
30 minutes.
DNA was extracted from DNA pellet.

2. DTAB (Dodecytrimethylammonium-bromide)
method

50 mg of the tissue was ground in 50mg of

liquid nitrogen. Then eppendorf tube
containing 700pi of hot extraction buffer
was taken, the ground tissue was added
and mixed rapidly, then incubated at 60 0C
for 30 minutes.
After chlolroform extraction, 1.7 volumes of
0.5% CTAB, 40mM NaCl was mixed at room
temperature and the DNA precipitated out
within 5 minutes, and at the end of DNA
extraction.

3. Guanidine-silica method

First of all, 50 mg of the tissue was

ground in 50mg of liquid nitrogen. Then
eppendorf tube containing 700pi of hot
extraction buffer was taken, the ground
tissue was added and mixed rapidly, then
incubated at 60oC for 30 minutes.
A silica particle suspension (10pi) was
added after phenol and chloroform
extractions and the given protocol used.

4. The DNA extraction Protocol of Doyle & Doyle (1990)

50 mg of the tissue was ground in 50mg of

liquid nitrogen. Then eppendorf tube
containing 700pi of hot extraction buffer
was taken, the ground tissue was added and
mixed rapidly, then incubated at 600C for 30
minutes.
After phenol and chloroform extractions, the
buffer was filtered and washed twice in
Microtone 30 tubes (Amicon, Berkeley), with
400 pi water. The DNA was then washed
from the membrane with 50 pi of water.

DNA CLONING

DNA cloning essentially involves seven

steps:
Selection of a host and vector
Synthesis of DNA of vector
Synthesis of DNA for cloning
Formation of recombinant DNA
Addition of recombinant DNA into host
Choice of organisms having recombinant
DNA
Seperation of clones with required DNA
sequences and biological properties.

DNA CLONING

Higuchi et al., (1984) and Piibo, (1985)

were the pioneers who successfully
cloned the ancient DNA by direct cloning
technique.
Cloning efficiency of the ancient DNA is
very low, therefore, it is impossible to
clone it directly by this technique.

POLYMERASE CHAIN REACTION (PCR)

To run a PCR, DNA polymerases, buffers

and bases of DNA, template DNA and
primers are artificially provided.
The primers recognize a particular part of
DNA and DNA polymerase adds bases to
the
elongating
strand
but
DNA
polymerase requires a primer to initiate
polymerization.

IMPORTANCE OF PCR

To amplify a very small amount of DNA

even from a single cell.
PCR is used in forensic science.
By using PCR, many copies of ancient
DNA can be produced, and the copied
DNA can be further used for DNA cloning.

DNA EXTRACTION FROM FOSSILIZED BONES

It is not easy to extract DNA preserved in

fossil bones because the ancient DNA is
either damaged or contaminated at the
site where natural mutation occurred.
Therefore, on running PCR the products
are contaminated due to deamination of
DNA.

DNA EXTRACTION FROM FOSSILIZED BONES

Weiner et al showed that a large amount

of DNA is present in inter-grown crystal
aggregates
that
cannot
be
disaggregated.
According to Salamon et al, to extract
DNA from the crystal aggregates, it
should
be
treated
with
sodium
hypochloride (NaOCl), a strong oxidant.

DNA EXTRACTION FROM FOSSILS IN ROCKS

In these fossils, the soft tissues, intracellular organelles as well as many

organic compounds like flavonoids as well
as steroids are also preserved.
The fossil containing rocks are crushed
scraped to mortar and ground in dry ice
until fine powder is formed, then DNA is
extracted from it.

DNA EXTRACTION FROM

FOSSILS IN ROCKS

Golenberg et al. (1990) , recovered and

analyzed leaf specimens from the early
Miocene fossil deposits at Clarkia, Idaho,
U.S.A.
DNA was extracted from leaf and
reproductive
materials,
after
their
remains were scratched from the shale
surface by using the extraction procedure
of Rogers et al (1985).

DNA EXTRACTION FROM

FOSSILS IN ROCKS

When gel electrophoresis was performed

and the extracts were stained with
ethidium bromide, only one of the ten
extracts contained large amount of high
molecular mass DNA.

DNA EXTRACTION FROM FOSSIL POLLEN OF Abies spp.

Pollen grains were washed crushed with

the help of pipette tip in an eppendorf
tube.
To an eppendorf tube, containing 30 ill of
mineral oil was incubated at 370C for 60
minutes.
The pollen grains were added and the
eppendorf tube was heated at 950C.
These pollens were considered as
template extract.

PROBLEMS FOR AMPLIFICATION OF FOSSIL DNA MATERIAL

Contaminating DNA

To remove the inhibiting effect of

contaminants (clay particles, secondary
metabolites) on DNA amplification, Paabo
(1990) suggested the addition of Bovine
Serum Albumin (BSA) to reduce the
inhibitory effect of these contaminants on
action of polymerases.

PROBLEMS FOR AMPLIFICATION OF FOSSIL DNA MATERIAL

Small
DNA

quantities

of

target

Polymerization will be successful only

when the target DNA is in large quantity.
The reason is that when PCR is run some
quantity of the DNA is denatured. So, if
there were small amount of target DNA
molecules then PCR will result in little or
n product.

PROBLEMS FOR AMPLIFICATION OF FOSSIL DNA MATERIAL

Specificity of primers
Much part of fossilized DNA

is degraded
therefore it is not possible to design a
specific primer sequence of desired
length.
Therefore, a series of primers should be
made in amplification experiments.

Detection of PCR inhibiting activities

In order to detect PCR inhibiting

activities, PCR cross-reactions were
carried out using 1 pi of each herbarium
DNA extract with 1 pi of a diluted positive
DNA extract from fresh leaves of Ilex
aquifolium as template.
If amplification does not occur it means
that inhibitors are present which interfere
in the amplification process.

REMOVAL OF POTENTIAL INHIBITING ACTIVITIES

Inhibitors effects can be avoided by:

Treating the DNA extracts with Polyclar AT
suspension
Changing PCR conditions i.e., increasing
amount of MgCl2 from 2mM to 6mM by
adding BSA (Bovine Serum Albumin).

CONCLUSION

DNA extraction is used to find out the

phylogenetic
relationships
among
different groups of plants. This can be
done by DNA cloning and by running PCR.
With the help of these processes, the
ancient DNA can be amplified and
compared with standard DNA sequences
to check the taxonomic and phylogenetic
relationships among different plant
groups.

REFERENCES

1. Banerjee, M. & Brown, T. A. (2004) J.

Archaeol. Sci. 31, 5963.
2. Carter, M. J., Milton, I. D., 1993: An
inexpensive and simple method for DNA
purifica tions on silica particles. - Nucl.
Acids Res. 21: 1044.
3. Cooper, A., Rambaut, A., Macaulay, V.,
Willerslev, E., Hansen, A. J. &
Stringer, C. (2001) Science 292, 1655
1656.

REFERENCES

4. Cooper A, Poinar HN. 2000. Ancient DNA: Do it

right or not at all. Science 289: 1139.
5. DeNiro, M. & Weiner, S. (1988) Geochim.
Cosmochim. Acta 52, 2197
2206.
6. Doyle, J. J., Dickson, E. E., 1987: Preservation of
plant samples for DNA restriction endonuclease
analysis. - Taxon 36: 715-722. - Doyle, J. L., 1990:
Isolation of plant DNA from fresh tissue. - Focus 12:
13-15.
7. Gilbert, M. T. P., Hansen, A. J., Willerslev, E.,
Rudbeck, L., Barnes, I., Lynnerup, N. & Cooper, A.
(2003) Am. J. Hum. Genet. 72, 4861.

REFERENCES

8. Gilbert, M. T. P., Willerslev, E., Hansen, A. J.,

Barnes, I., Rudbeck, L.,Lynnerup, N. & Cooper, A.
(2003) Am. J. Hum. Genet. 72, 3247.
9. Gilbert, M. T. P., Rudbeck, L., Willerslev, E.,
Hansen, A. J., Smith, C. I.,
Penkman, K. E. H., Prangenberg, K., Nielsen-Marsh
C. M., Jans, M. E., Arthur,
P., et al. (2005) J. Arch. Sci. 32, 785793.
10. Golenberg, E. M., Giannassi, D. E., Clegg, M. T.,
Smiley, C.J., Durbin, M., Henderson, D. & Zurawski,
G. 1990 Chloroplast DNA sequence from a Miocene
Magnolia species. Nature, Lond. 344, 656-658.