DNA extraction

The first isolat ion of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher.[1]
Current ly it is a rout ine procedure in molecular biology or forensic analyses. For t he chemical
met hod, t here are many different kit s used for ext ract ion, and select ing t he correct one will
save t ime on kit opt imizat ion and ext ract ion procedures. PCR sensit ivit y det ect ion is
considered t o show t he variat ion bet ween t he commercial kit s.[2]

Basic procedure

Cells which are t o be st udied need t o be collect ed.

Breaking t he cell membranes open t o expose t he DNA along wit h t he cyt oplasm wit hin
(cell lysis).
Lipids from t he cell membrane and t he nucleus are broken down wit h det ergent s and
surfact ant s.

Breaking down prot eins by adding a prot ease (opt ional).

Breaking down RNA by adding an RNase (opt ional).

The solut ion is t reat ed wit h a concent rat ed salt solut ion (saline) t o make debris such as
broken prot eins, lipids and RNA clump t oget her.

Cent rifugat ion of t he solut ion, which separat es t he clumped cellular debris from t he DNA.

DNA purificat ion from det ergent s, prot eins, salt s and reagent s used during t he cell lysis
st ep. The most commonly used procedures are:
Et hanol precipit at ion usually by ice-cold et hanol or isopropanol. Since DNA is insoluble
in t hese alcohols, it will aggregat e t oget her, giving a pellet upon cent rifugat ion.
Precipit at ion of DNA is improved by increasing of ionic st rengt h, usually by adding
sodium acet at e.
 Phenol–chloroform ext ract ion in which phenol denat ures prot eins in t he sample. Aft er
cent rifugat ion of t he sample, denat ured prot eins st ay in t he organic phase while t he
aqueous phase cont aining nucleic acid is mixed wit h chloroform t o remove phenol
residues from t he solut ion.

Minicolumn purificat ion t hat relies on t he fact t hat t he nucleic acids may bind
(adsorpt ion) t o t he solid phase (silica or ot her) depending on t he pH and t he salt
concent rat ion of t he buffer.

Cellular and hist one prot eins bound t o t he DNA can be removed eit her by adding a prot ease or
by having precipit at ed t he prot eins wit h sodium or ammonium acet at e, or ext ract ed t hem
wit h a phenol-chloroform mixt ure prior t o t he DNA-precipit at ion.

Aft er isolat ion, t he DNA is dissolved in a slight ly alkaline buffer, usually in a TE buffer, or in
ult ra-pure wat er.

Method selection

Some of t he most common DNA ext ract ion met hods include organic ext ract ion, Chelex
ext ract ion, and solid phase ext ract ion.[3] These met hods consist ent ly yield isolat ed DNA, but
t hey differ in bot h t he qualit y and t he quant it y of DNA yielded. When select ing a DNA
ext ract ion met hod, t here are mult iple fact ors t o consider, including cost , t ime, safet y, and risk
of cont aminat ion.

Organic ext ract ion involves t he addit ion of and incubat ion in mult iple different chemical
solut ions;[3] including a lysis st ep, a phenol chloroform ext ract ion, an et hanol precipit at ion, and
washing st eps. Organic ext ract ion is oft en used in laborat ories because it is cheap, and it
yields large quant it ies of pure DNA. Though it is easy, t here are many st eps involved, and it
t akes longer t han ot her met hods. It also involves t he unfavorable use of t he t oxic chemicals
phenol and chloroform, and t here is an increased risk of cont aminat ion due t o t ransferring t he
DNA bet ween mult iple t ubes.[4] Several prot ocols based on organic ext ract ion of DNA were
effect ively developed decades ago,[5] t hough improved and more pract ical versions of t hese
prot ocols have also been developed and published in t he last years.[6]

Chelex ext ract ion met hod involves adding t he Chelex resin t o t he sample, boiling t he solut ion,
t hen vort exing and cent rifuging it . The cellular mat erials bind t o t he Chelex beads, while t he
DNA is available in t he supernat ant .[4] The Chelex met hod is much fast er and simpler t han
organic ext ract ion, and it only requires one t ube, which decreases t he risk of DNA
cont aminat ion. Unfort unat ely, Chelex ext ract ion does not yield as much quant it y and t he DNA
yielded is single-st randed, which means it can only be used for PCR-based analyses and not
for RFLP.[4]

Solid phase ext ract ion such as using a spin-column based ext ract ion met hod t akes advant age
of t he fact t hat DNA binds t o silica. The sample cont aining DNA is added t o a column
cont aining a silica gel or silica beads and chaot ropic salt s. The chaot ropic salt s disrupt t he
hydrogen bonding bet ween st rands and facilit at e binding of t he DNA t o silica by causing t he
nucleic acids t o become hydrophobic. This exposes t he phosphat e residues so t hey are
available for adsorpt ion.[7] The DNA binds t o t he silica, while t he rest of t he solut ion is
washed out using et hanol t o remove chaot ropic salt s and ot her unnecessary const it uent s.[3]
The DNA can t hen be rehydrat ed wit h aqueous low salt solut ions allowing for elut ion of t he
DNA from t he beads.

This met hod yields high-qualit y, largely double-st randed DNA which can be used for bot h PCR
and RFLP analysis. This procedure can be aut omat ed[4] and has a high t hroughput , alt hough
lower t han t he phenol-chloroform met hod. This is a one-st ep met hod i.e t he ent ire procedure
is complet ed in one t ube. This lowers t he risk of cont aminat ion making it very useful for
forensic ext ract ion of DNA. Mult iple solid phase ext ract ion commercial kit s are manufact ured
and market ed by different companies; t he only problem is t hat t hey are more expensive t han
organic ext ract ion or Chelex ext ract ion.

Special types

Specific t echniques must be chosen for isolat ion of DNA from some samples. Typical
samples wit h complicat ed DNA isolat ion are:

archaeological samples cont aining part ially degraded DNA, see ancient DNA[8]

samples cont aining inhibit ors of subsequent analysis procedures, most not ably inhibit ors of
PCR, such as humic acid from soil, indigo and ot her fabric dyes or haemoglobin in blood

samples from microorganisms wit h t hick cellular wall, for example yeast

samples cont aining mixed DNA from mult iple sources

Ext rachromosomal DNA is generally easy t o isolat e, especially plasmids may be easily
isolat ed by cell lysis followed by precipit at ion of prot eins, which t raps chromosomal DNA in
insoluble fract ion and aft er cent rifugat ion, plasmid DNA can be purified from soluble fract ion.

A Hirt DNA Ext ract ion is an isolat ion of all ext rachromosomal DNA in a mammalian cell. The
Hirt ext ract ion process get s rid of t he high molecular weight nuclear DNA, leaving only low
molecular weight mit ochondrial DNA and any viral episomes present in t he cell.
 Detection of DNA

A diphenylamine (DPA) indicat or will confirm t he presence of DNA. This procedure involves
chemical hydrolysis of DNA: when heat ed (e.g. ≥95 °C) in acid, t he react ion requires a
deoxyribose sugar and t herefore is specific for DNA. Under t hese condit ions, t he 2-
deoxyribose is convert ed t o w-hydroxylevulinyl aldehyde, which react s wit h t he compound,
diphenylamine, t o produce a blue-colored compound. DNA concent rat ion can be det ermined
measuring t he int ensit y of absorbance of t he solut ion at t he 600 nm wit h a
spect rophot omet er and comparing t o a st andard curve of known DNA concent rat ions.

Measuring t he int ensit y of absorbance of t he DNA solut ion at wavelengt hs 260 nm and 280
nm is used as a measure of DNA purit y. DNA can be quant ified by cut t ing t he DNA wit h a
rest rict ion enzyme, running it on an agarose gel, st aining wit h et hidium bromide (Et Br) or a
different st ain and comparing t he int ensit y of t he DNA wit h a DNA marker of known
concent rat ion.

Using t he Sout hern blot t echnique, t his quant ified DNA can be isolat ed and examined furt her
using PCR and RFLP analysis. These procedures allow different iat ion of t he repeat ed
sequences wit hin t he genome. It is t hese t echniques which forensic scient ist s use for
comparison, ident ificat ion, and analysis.

High-molecular-weight DNA extraction method

In t his met hod, plant nuclei are isolat ed by physically grinding t issues and reconst it ut ing t he
int act nuclei in a unique Nuclear Isolat ion Buffer (NIB). The plast id DNAs are released from
organelles and eliminat ed wit h an osmot ic buffer by washing and cent rifugat ion. The purified
nuclei are t hen lysed and furt her cleaned by organic ext ract ion, and t he genomic DNA is
precipit at ed wit h a high concent rat ion of CTAB. The highly pure, high molecular weight gDNA
is ext ract ed from t he nuclei, dissolved in a high pH buffer, allowing for st able long-t erm
st orage.[9]

See also

Boom met hod

DNA fingerprint ing

DNA sequencing

DNA st ruct ure

Et hanol precipit at ion

Plasmid preparat ion

Polymerase chain react ion

SCODA DNA purificat ion

References

1. Dahm R (January 2008). "Discovering DNA: Friedrich Miescher and the early years of nucleic acid
research". Human Genetics. 122 (6): 565–81. doi:10.1007/s00439-007-0433-0 (https://doi.org/10.1
007%2Fs00439-007-0433-0) . PMID 17901982 (https://pubmed.ncbi.nlm.nih.gov/17901982) .
S2CID 915930 (https://api.semanticscholar.org/CorpusID:915930) .

2. Yoshikawa H, Dogruman-Al F, Dogruman-Ai F, Turk S, Kustimur S, Balaban N, Sultan N (October

2011). "Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes
from fecal samples". Parasitology Research. 109 (4): 1045–50. doi:10.1007/s00436-011-2342-3 (htt
ps://doi.org/10.1007%2Fs00436-011-2342-3) . PMID 21499752 (https://pubmed.ncbi.nlm.nih.gov/
21499752) . S2CID 37191780 (https://api.semanticscholar.org/CorpusID:37191780) .

3. Elkins KM (2013). "DNA Extraction". Forensic DNA Biology. pp. 39–52. doi:10.1016/B978-0-12-

394585-3.00004-3 (https://doi.org/10.1016%2FB978-0-12-394585-3.00004-3) .
ISBN 9780123945853.

4. Butler JM (2005). Forensic DNA typing : biology, technology, and genetics of STR markers (2nd ed.).
Amsterdam: Elsevier Academic Press. ISBN 9780080470610. OCLC 123448124 (https://www.worldc
at.org/oclc/123448124) .

5. Marmur, J. (1961). "A procedure for the isolation of deoxyribonucleic acid from micro-organisms".
Journal of Molecular Biology. 3 (2): 208–IN1. doi:10.1016/S0022-2836(61)80047-8 (https://doi.org/
10.1016%2FS0022-2836%2861%2980047-8) .

6. Salvà-Serra F, Svensson-Stadler L, Busquets A, Jaén-Luchoro D, Karlsson R, Moore ER, Gomila M

(2018). "A protocol for extraction and purification of high-quality and quantity bacterial DNA
applicable for genome sequencing: A modified version of the Marmur procedure" (https://doi.org/10.
1038%2Fprotex.2018.084) . Protocol Exchange. doi:10.1038/protex.2018.084 (https://doi.org/10.
1038%2Fprotex.2018.084) .

7. Li, Richard (11 March 2015). Forensic biology (2nd ed.). Boca Raton. ISBN 978-1439889725.
OCLC 907517669 (https://www.worldcat.org/oclc/907517669) .

8. Pääbo S (March 1989). "Ancient DNA: extraction, characterization, molecular cloning, and enzymatic
amplification" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC286820) . Proceedings of the
National Academy of Sciences of the United States of America. 86 (6): 1939–43.
Bibcode:1989PNAS...86.1939P (https://ui.adsabs.harvard.edu/abs/1989PNAS...86.1939P) .
doi:10.1073/pnas.86.6.1939 (https://doi.org/10.1073%2Fpnas.86.6.1939) . PMC 286820 (https://
 www.ncbi.nlm.nih.gov/pmc/articles/PMC286820) . PMID 2928314 (https://pubmed.ncbi.nlm.nih.g
ov/2928314) .

9. Li, Zhigang; Parris, Stephen; Saski, Christopher A. (2020). "A simple plant high-molecular-weight DNA
extraction method suitable for single-molecule technologies" (https://www.ncbi.nlm.nih.gov/pmc/arti
cles/PMC7071634) . Plant Methods. 16: 38. doi:10.1186/s13007-020-00579-4 (https://doi.org/10.
1186%2Fs13007-020-00579-4) . ISSN 1746-4811 (https://www.worldcat.org/issn/1746-4811) .
PMC 7071634 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071634) . PMID 32190102 (http
s://pubmed.ncbi.nlm.nih.gov/32190102) . Text was copied from this source, which is
available under a Creative Commons Attribution 4.0 International License (https://creativecommon
s.org/licenses/by/4.0/) .

Further reading

Sambrook, Michael R. Green, Joseph. Molecular Cloning. (4t h ed. ed.). Cold Spring Harbor,
N.Y.: Cold Spring Harbor Laborat ory Pr. ISBN 1936113422.

Forensic Biology, Richard Li, (2015) Boca Rat on : CRC Press, Taylor & Francis Group.
ISBN 9781439889701

External links

How t o ext ract DNA from anyt hing living (ht t p://learn.genet ics.ut ah.edu/cont ent /labs/ext r
act ion/howt o/)

DNA Ext ract ion Virt ual Lab (ht t p://learn.genet ics.ut ah.edu/cont ent /labs/ext ract ion/)

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