PCR methods & point mutations
n.panjwani at lshtm.ac.uk
Mon Jul 17 06:05:43 EST 1995
Francesc Xavier Lopez-Labrador <100647.73 at compuserve.com> wrote:
> Looking for information of PCR methods (different that ARMS or COP)to
> detect viral point mutations.
I would suggest SSCP (Single Stranded Conformation Polymorphism). Hot
PCR products amplified from a (poentially mutable) portion of the
viral genome are heated to denature, and immediately run on a glycerol
containing polyacrylamide gel, against a known wild type PCR product
amplified with the same primers. Point mutations will show up as
shifts in mobility compared to the wild type, as well as the
appearance or disappearance of bands compared to the wild type pattern.
The banding pattern is a reflection of the various stable conformations
of the single strand, and the shifts are based on alternative
conformations assumed by a non wild type fragment, which are brought
about at the scale of a single base difference.
This technique has been used in the past with Human Papilloma Viruses
and others (see below), and with great success to detect point
mutations in hypermutable exons (5-8) of p53 gene. Can be used in
conjunction with RT-PCR for RNA viruses (see below)
Note: Cold PCR should be done first to rule out large scale genomic re-
organisation, as this would complicate interpretation of the SSCP.
See the following two abstracts to get an idea of application in
TI: Posttransplantation lymphoproliferative disorders frequently
contain type A and not type B
AU: Frank-D; Cesarman-E; Liu-YF; Michler-RE; Knowles-DM
AD: Department of Pathology, College of Physicians and Surgeons of
Columbia University, New York, NY, 10021.
SO: Blood. 1995 Mar 1; 85(5): 1396-403
AB: Two families of Epstein-Barr virus (EBV), type A and type B, have
been defined on the basis of sequence divergence in the EBNA-2 gene.
Type A EBV immortalizes B cells more efficiently in vitro and infects
immunocompetent individuals more commonly than type B EBV. However,
increased rates of infection by type B EBV are seen in
immunocompromised hosts and in many lymphoid neoplasms associated
with immunocompromise. The posttransplantation lymphoproliferative
disorders (PT-LPDs) are a heterogeneous group of B-cell neoplasms
that arise in the setting of immunosuppressive therapy, and are
associated with EBV infection. Whether type A and/or type B EBV
are associated with PT-LPDs is unknown. Therefore, we investigated
27 PT-LPD lesions from 22 solid-organ transplant recipients by
polymerase chain reaction (PCR) at the EBNA-2 and EBNA-3c loci
to detect sequence deletions that distinguish the two EBV families.
Another locus, EBER, was examined by single-strand conformation
polymorphism analysis (SSCP), in conjunction with direct sequencing
in selected cases. Type A EBV was found in 24 of 27 cases (89%) as
seen by amplification of the EBNA-2 and EBNA-3c regions.
Four different EBER polymorphisms were detected, confirming the
presence of different type A EBV isolates among these cases.
Three cases were negative for infection by EBV. Surprisingly,
despite the immunocompromised state of the hosts, none of the 27
PT-LPD lesions harbored type B EBV. Thus, although type B EBV may
commonly infect peripheral blood lymphocytes in immunocompromised
individuals, they do not appear to induce readily PT-LPD formation.
------------------------------- 2 -----------------------------------
TI: Evolution and selection of hepatitis C virus variants in patients
with chronic hepatitis C.
AU: Kurosaki-M; Enomoto-N; Marumo-F; Sato-C
AD: Second Department of Internal Medicine, Faculty of Medicine,
Tokyo Medical and Dental University, Japan.
SO: Virology. 1994 Nov 15; 205(1): 161-9
AB: It has been shown that hepatitis C virus (HCV) populations in vivo
are composed of different but highly homologous HCV genomes
(quasispecies) as shown in the hypervariable region (HVR) that exists
in the N-terminal of the envelope 2 gene of HCV, and that the
predominant sequence of the HVR of HCV genomes changes rapidly over
time. To further investigate genetic backgrounds of the change in the
HVR of HCV genomes, 45 plasma samples serially obtained from nine
patients with chronic hepatitis C were studied using population-based
analyses. Total RNA was recovered and the envelope gene containing
the HVR was amplified by the reverse transcription and nested
polymerase chain reaction. The amplified cDNA was examined by the
single strand conformation polymorphism (SSCP) analysis.
Furthermore, 43 HCV sequences, separated by the SSCP analysis from
three patients were determined by the dideoxy chain termination
method, and the phylogenetic analysis was performed using the
neighbor joining method. The SSCP analysis demonstrated that HCV
population within each individual were composed of 1 to 6
quasispecies. These quasispecies populations in vivo changed
sequentially in eight of nine patients. Gradual selections of
coexisting quasispecies were observed over 6- to 18-month periods in
three patients, whereas complete replacements of previous
quasispecies by new quasispecies were repeatedly observed over
few-month intervals in five patients. The phylogenetic analysis on
these quasispecies revealed the continuous accumulation of mutations
in two patients and discontinuous appearance of evolutionarily
distant quasispecies in one patient. These results indicate that HCV
genomes in vivo form quasispecies populations, and that these
quasispecies populations change during the natural course of chronic
infection. Genetic mechanisms underlining the change of the HVR of
HCV genome appear to be either continuous accumulation of mutations
or selective overgrowth of preexisting minor variants from the large
spectrum of quasispecies populations.
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