Estudo_sob_orientação_do_professor_Eduardo_Ramalho_que_foi_submetido_em_2002_e_publicado_em_2005_numa_revista_internacional..pdf
Documento PDF (88.9KB)

Exibir conteúdo Baixar

                    Made with Xodo PDF Reader and Editor

37

Vol.48, n. 1 : pp. 37-40, January 2005
ISSN 1516-8913 Printed in Brazil

BRAZILIAN ARCHIVES OF
BIOLOGY AND TECHNOLOGY
A N

I N T E R N A T I O N A L

J O U R N A L

High Quality DNA from Human Papillomavirus (HPV) for
PCR/RFLPs
Denise Wanderlei-Silva1*, Mariana Nobre3, Rosa Silva Gonzaga2, Luciana Silva Viana2 and
Eduardo Ramalho Neto2
1

Departamento de Patologia; CCBi; Universidade Federal de Alagoas - UFAL; 57010-020; dmws@ofm.com.br;
Maceió - AL - Brazil. 2 Laboratório de Genética Molecular; Genômica e Proteômica - GEMPRO; CECA;
Universidade Federal de Alagoas - UFAL; 57100-000; Maceió - AL - Brazil. 3 Seção de Patologia Cervical,
Hospital Universitário; Universidade Federal de Alagoas - UFAL; 57072-970; Maceió - AL - Brazil

ABSTRACT
The analysis of DNA in clinical samples for a secure diagnostic has become indispensable nowadays. Techniques
approaching isolation of high molecular weigth DNA of HPV could lead to efficient amplification and early clinical
diagnosis of the virus DNA by PCR (polymerase chain reaction). We describe a fast, non-toxical, efficient and
cheap method for DNA isolation of human papilloma virus (HPV) from cervical smears using guanidine (DNAzol
solution). A 450 bp DNA band correponding to the late region (L1) of the virus genome was detected by PCR,
showing that the DNAzol extraction soluction generated a good viral DNA yield. The electrophoretic pattern after
digestion with restriction endonucleases (RFLPs/PCR) revealed the predominance of HPV-16 and HPV-33 in the
samples from the State of Alagoas, Brazil.
Key words: Human papillomavirus, DNA purification, PCR, RFLP

INTRODUCTION
More than 100 different types of human
papillomavirus (HPV) have now been identified
by molecular techniques of which almost 30% are
mucosotropic (Harnish et al. 1999). They can
infect the anogenital tract and contain DNA of
high-risk type which is strongly associated with
cervical carcinoma (Villa 1997, Walboomers et al.
1999, Wilczynski et al. 1988). Assessment of
samples for HPV infection with PCR amplification
is important for early detection of initial lesions
and virus typing.
The isolation of DNA is an essential step for any
technique in Molecular Biology. There have been
several recent reports describing different
*

protocols for viral DNA/RNA suitable for PCR
amplification (Van der Brule et. al. 1990, ElufNeto et al. 1994). The majority of them uses
harzadous chemicals, such as phenol and
chloroform (Wilczynski et al. 1988), which are
laborious, expensive and time consuming. They
also involve several steps and are time-consuming.
We describe a modification of a protocol using
guanidine detergent (DNAzol) for purification of
HPV DNA suitable for PCR amplification. The
main objective of this work was to develop a fast,
high reliable and low cost protocol for isolation of
quality woman HPV DNA from cervical samples.

Author for correspondence

Brazilian Archives of Biology and Technology

Made with Xodo PDF Reader and Editor

38

Wanderlei-Silva, D. et al.

MATERIALS AND METHODS
Sample preparation
A total of 100 cervical smears samples were
collected from women attending the Cervical
Pathology service of the Federal University of
Alagoas Hospital and from private clinics of the
State of Alagoas by sampling cells from
endocervix and ectocervix with a brush. We
selected women with history of any previous
suggestive
Papanicolaou
(Pap),
cervical
intraepithelial neoplasia (CIN) I, II, and III. The
material was placed into Eppendorf tubes
containing 500µL of DNA extraction solution
(DNAzol – Life Technologies) and after 4 to 5
days were sent to the Molecular Genetics
Laboratory of the Federal University of Alagoas.
DNA isolation
Genomic DNA isolation was performed by
modifying a protocol based on guanidine
detergent, the DNAzol solution. The tubes with the
samples were inverted several times and DNA
precipitation was done by adding 250µL ice-cold
isopropanol. The tubes were centrifuged for 1 to 2
min at 4300xg to collect the DNA pellets and the
supernatant was carefully discarded. The pellets
were washed with 800µL of 95% ethanol by
inverting tubes three to six times. The ethanol was
removed and tubes were stored for 15 minutes in
order to dry the DNA at room temperature. The
pellets were ressuspended in 100µL of 8mM
NaOH and stored at 4°C.
PCR amplification and restriction analysis
PCR amplification of HPV L1 region was
accomplished using primers MY09 (5’CGTCCMAARGGAWACTGATC-3’) and MY11
(5’-GCMCAGGGWCATAAYAATGG-3’). The
co-amplification of the human β-globin gene
(approximately 268 bp) was performed by using
primers GH20 (5’-GAAGAGCCAAGGACAGG
TAC-3’) and PC04 (5’-CAACTTCATCCACGTT
CACC-3’), according to Bauer et al. (1992). Two
types of controls were also included in each
reaction series: ‘no-DNA’ and ‘HPV-positive
DNA’.
All clinical samples employed in this study were
positive for β-globin gene after PCR reaction with
globin primers. PCR amplification reactions were
performed in 100µL, with the following
components and final concentrations: 1X Taq

buffer, 200µM deoxiribonucleoside triphosphates
(dNTPs), 1.5mM MgCl2, 1.0µM of primers
MY09/MY11 and 1.0µM of primers GH20/PC04,
2U Taq DNA polimerase, DNA template,
20ng/100µL. PCR reactions were subjected to 35
cycles of amplification on a thermal cycler
(Hybaid) using the following program:
denaturation at 94ºC for 30 sec, annealing at 55ºC
for 1 min, extension at 72ºC for 1 min, and final
extension at 72ºC for 8 min. Amplification
products were separated by electrophoreses in
1.5% agarose gels, stained with ethidium bromide
and photographed under UV light using a Kodak
digital system.
The results of amplification reactions were
assessed by scoring sample as either HPV-positive
or -negative. Viral PCR products of 450 pb were
cleaved with the restriction enzymes: BamHI,
Ddel, HaeIII, HinfI, PstI, RsaI, and Sau3AI (Life
Technologies), according to Villa (1997). Digested
fragments were detected by 8% acrylamide
electrophoresis and stained with silver nitrate
(Sanguinetti et al., 1994).

RESULTS
DNAzol extraction solution yielded genomic DNA
of high molecular weight with fragments of
about 23 kb. PCR amplification of HPV late (L1)
region accomplished using primers MY09 and
MY11 generated fragments of approximately
450 pb (Fig. 1).
Approximately 40% of the samples were HPV
positive. Amplimers were separated in 1.5%
agarose gel (Fig. 1) by electrophoresis (4V.cm-1)
and stained with ethidium bromide. The products
of the HPV positive samples were cleaved with the
restriction enzymes (RFLPs/PCR) generatig
physical maps that could be compared to existing
HPV maps, allowing identification of different
viral types, and consequently their exclusion or
inclusion in high-risk types. The electrophoretic
pattern after digestion revealed the predominance
of HPV-33 (Fig. 2) and HPV-16 (Fig. 3) in the
samples analysed.

Brazilian Archives of Biology and Technology

Made with Xodo PDF Reader and Editor

High Quality DNA from Human Papillomavirus (HPV) for PCR/RFLPs

L

1

2

3

4

5

6

7

8

2072bp

39

proteinase K, and phenol extractions (Wilczynski et
al. 1988) or unecessary steps such as freezing
followed by heating at high temperatures of cell
suspension before PCR (Van der Brule et al. 1990).

100bp
L1

L

9

10

w/d

BamHI DdeI

HaeIII HinfI

PstI

RsaI Sau3AI

11

2072bp
100bp

Figure 1 - Detection of HPV DNA from clinical samples
amplified with primers MY09 and MY11
(lanes 3, 5 and 7). HPV-negative is shown in
lane 1. Controls consist of no target DNA (11)
and HPV-positive DNA (9). β-globin DNA,
the reaction control was always amplified
(lanes 2, 4, 6, 8 and 10). L= 100 bp ladder
(Life Technologies-Gibco).

L1

w/d

BamHI DdeI HaeIII HinfI

PstI

RsaI Sau3AI

Figure 2 - 8% silver stained acrylamide gel showing
RFLP pattern of the HPV type 33 after
digestion with the six restriction enzymes.
L1= 100pb ladder (Life TechnologiesGibco). w/d= PCR amplified fragment
without digestion (~450bp). Electrophoresis:
5v.cm-1.

DISCUSSION
Molecular studies of HPV have been carried out
using different methodologies for viral DNA
isolation from clinical samples. Typical protocols
use phosphate-buffered saline (PBS) (Eluf-Neto
et al. 1994), sodium-dodecyl-sulfate (SDS),

Figure 3 - 8% silver stained acrylamide gel showing
RFLP pattern of the HPV type 16 after
digestion with the six restriction enzymes.
L1= 100pb ladder (Life TechnologiesGibco). w/d= PCR amplified fragment
without
digestion
(~450bp).
Electrophoresis: 5v.cm-1.

Clinical samples from smears might contain blood
or endogenous enzymes able to prevent PCR
reaction. Furthermore, Taq DNA polimerase can
be inhibited by chemicals added to buffers to
deactivate infectious agents present in blood and
clinical smears (Gravitt and Manos 1992).
Despite the use of traditional protocols, kits for
genomic DNA extraction are becoming very
popular, mostly because they are ready to use and
contain non toxic reagents, such as DNAzol
Reagent, a genomic DNA isolation reagent which
uses a guanidine-detergent lysing solution for
selective precipitation of DNA from cell lysate. It
is recommended for purification of DNA from
solid and liquid samples from animal, yeast,
bacteria and plant sources.
DNAzol extraction solution allowed celular lysis
and DNA precipitation in simultaneous steps. An
advantage of the solution is to allow samples to be
stored for longer periods of time without
compromising DNA integrity. It also produced
good DNA suitable for HPV detection and
identification. The method is simple, fast (20
minutes) and generates DNA of high molecular
weight. Another advantage is that no griding is
necessary to obtain sufficient DNA.
The efficacy of the protocol was determined by
PCR amplifying a fragment corresponding to the
L1 region of the virus. The 450 bp fragments

Brazilian Archives of Biology and Technology

Made with Xodo PDF Reader and Editor

40

Wanderlei-Silva, D. et al.

indicating the presence of the virus in the samples
were readly used for assignment of HPV type by
restriction enzyme pattern (RFLP). The physical
maps generated allowed identification of different
viral types, according to Bernard et al. (1994), and
consequently their exclusion or inclusion in highrisk types. Human papilloma virus has been
classified in more than 100 types according to its
malignat potential assessed by molecular
techniques (Harnish et al. 1999). The most
common high-risk types HPVs are 16, 18, 31, 33,
35, 51, and 52, which have been associated with
CIN II, CIN III and invasive cervical cancer (ElufNeto et al. 1994). Our preliminar data show that
the predominant types in the State of Alagoas are
HPV 16 and HPV 33, both of them presenting
high oncogenic potential. Our investigation further
support the role of these high-risk types HPVs in
the development of cervical neoplasia.

ACKNOWLEDGEMENTS
This work was financially supported by CNPq,
Brazilian National Council for Research and
Development and FAPEAL, Alagoas State
Research Foundation, Brazil.

RESUMO
A detecção de DNA em amostras clínicas visando
um diagnóstico mais seguro vem se tornando uma
prática comum em laboratórios de análise clínica.
Metodologias que objetivem o isolamento de DNA
de alto peso molecular de HPV podem levar a uma
amplificação precisa e diagnose precoce do DNA
do vírus por PCR (reação de polimerase em cadeia).
Nós descrevemos um método para o isolamento do
DNA do vírus do papiloma humano de amostras
cervicais utilizando o detergente guanidina (solução
DNAzol). O método foi rápido, não-tóxico e
eficiente. Uma banda de DNA de 450 pb
correspondente à região tardia (L1) do genoma viral
foi detectada por PCR, mostrando que a extração
com DNAzol gerou quantidade suficiente de DNA
para análise. O padrão eletroforético, após digestão
com endonucleases de restrição (RFLPs/PCR),
revelou predominância de HPV 16 e HPV-33 nas
amostras no Estado de Alagoas, Brasil.

REFERENCES
Bernard, H. U.; Chan, S. Y.; Manos, M. M.; Ong, C. K.;
Villa, L. L.; Delius, H.; Peyton, C. L.; Bauer, H. M.
and Wheeler, C. M. (1994), Identification and
assessment
of known
and novel
human
papillomaviruses by Polymerase Chain Reaction
amplification,
restriction
fragment
length
polymorphisms, nucleotide sequence, and phylogenetic
algorithms. J. Infect. Dis., 170, 1077-1085.
Bauer, H. M.; Greer, C. E. and Manos, M. M. (1992)
Detection of genital human papillomavirus
infection using PCR. In: Herrington, C. S. and
McGee, J. O. D. (Eds.). Diagnostic molecular
pathology: a practical approach. Oxford : Oxford
University Press. pp. 131-152.
Eluf-Neto, J.; Booth, M.; Muñoz, N.; Bosch, F. X.;
Meijer, C. J. L. M. and Walboomers, J. M. M. (1994),
Human papillomavirus and invasive cervical cancer
in Brazil. J. Cancer, 69, 114-119.
Gravitt, P. E. and Manos, M. M. (1992), Polymerase
chain reaction - based methods for the detection of
human papilomavirus DNA. The Epidemiology of
cervical cancer and human Papilomavirus. IARC.
pp. 121-133.
Harnish, D. G.; Belland, L. M.; Scheid, E. E. and
Rohan, T. E. (1999), Evaluation of human
papillomavirus-consensus primers for HPV detection
by the polymerase chain reaction. Molecular and
Cellular Probes, 13, 9-21.
Sanguinetti, C. J.; Neto, E. D. and Simpson, A. J. G.
(1994), Rapid silver staining and recovery of PCR
products separated on polyacrylamide gels.
BioTechniques, 16, 915-919.
Van der Brule, A. J. C.; Meijer, C. J. L. M.; Bakels, V.;
Kenemans, P. and Walboomers, J. M. M. (1990),
Rapid detection oh human papillomavirus in cervical
scrapes by combined general primer-mediated and
type-specific polymerase chain reaction. J. Clin.
Microbiol, 28, 2739-2743.
Villa, L. L. (1997), Human Papillomaviruses and
Cervical Cancer. In: Advances in Cancer Research.
Academic Press. pp. 322-341.
Walboomers, J. M. M.; Jacobs, M. V. and Manos,
M. M. et al. (1999), Human papillomavirus is a
necessary cause for invasive cervical cancer
worldwide. J. Pathol, 189, 12-19.
Wilczynski, S. P.; Bergen, S., W;alker, J.; Liao, S. Y.
and Pearlman, L. F. (1988), Human papillomaviruses
and cervical cancer: Analysis of hystopathologic
features associated with different viral types. Human
Pathology, 19, 697-704.

Brazilian Archives of Biology and Technology

Received: February 07, 2002;
Revised: September 25, 2003;
Accepted: August 30, 2004.