Nova Publishers
My Account Nova Publishers Shopping Cart
HomeBooksSeriesJournalsReference CollectionseBooksInformationSalesImprintsFor Authors
  Top » Catalog » Books » Biology » Genetics » New Research on DNA Damage Chapters » My Account  |  Cart Contents  |  Checkout   
Quick Find
Use keywords to find the product you are looking for.
Advanced Search
What's New? more
Advances in Materials Science Research. Volume 32
Shopping Cart more
0 items
Shipping & Returns
Privacy Notice
Conditions of Use
Contact Us
Notifications more
NotificationsNotify me of updates to Mutation Detection in the TP53 Gene: Alternatives in Point Mutation Technologies (pp. 343-345)
Tell A Friend
Tell someone you know about this product.
Mutation Detection in the TP53 Gene: Alternatives in Point Mutation Technologies (pp. 343-345) $100.00
Authors:  (Rangel-Lòpez Angélica, Unidad de Investigacion Médica en Enfermedades Oncològicas, Hospital de Oncologìa, Centro Médico Nacional S XXI, laboratorio de Tecnologia del DNA, Departamento de Bioquimica, Escuela Nacional de Ciencias Biològicas, Méndez-Tenorio Alfonso, Laboratorio de Tecnologìa del DNA, Departamento de Bioquìmica, Escuela Nacional de Ciencias Biològicas, Salcedo Mauricio, Unidad de Investigaciòn Médica en Enfermedades Oncològicas, Hospital de Oncologia, México)
One of the major challenges involved mainly in searching of point mutations for clinical
relevance is the technology used; in particular for cancer research we will focus on the gains
or loss-of-suppression function, in cancer genes as RET, TP53, RAS, etc. TP53 has been
used as an excellent model for point mutation detection, because of its more than 20,000
different mutations this gene is the most frequently found in many human cancers.
Furthermore, there are uncommon somatic and germline mutations that might be related
to specific cancers or predispositions. In particular case, the precise nature of the TP53
mutation presents both challenges and opportunities for alternate treatment strategies in
specific cancers. These highlight the clinical need to accurately identify often unknown
inherited aberrations or infrequently represented mutations in mixed populations of DNA
Different polymorphisms or mutation detection technologies have been developed to
identify known changes: these include DNA microarrays, the polymerase chain
reaction/ligase detection reaction (PCR/LDR), now used in combination with the universal
DNA microarray and primer extension assays. On the other hand, the technologies used for
unknown mutations: hybridization analysis using high-density oligonucleotide arrays,
denaturing high-performance liquid chromatography (DHPLC), capillary electrophoresisbased
single strand conformation polymorphism (CE-SSCP), denaturing gradient gel
electrophoresis (DGGE) and heteroduplex analysis (HA). Finally, dideoxy-sequencing has
difficulty in detecting heterozygous mutations, and is of limited utility in the analysis of solid
tumors where mutant DNA may represent as little as 15% of the total. Some other methods
include in vitro transcription/translation-based approaches, chemical and enzymatic mismatch
cleavage detection (e.g. Cleavase, RNase, T4 endonuclease VII, MutS enzymes and CEL I).
In vitro mismatch cleavage methods encounter variability in signal intensity compared with
background bands.
Hybridization analysis using low-density oligonucleotide arrays for unknown mutations,
certainly might have the potential for custom fabrication and the detection of all desired
features and could conveniently provide reliable results and decrease production cost
considerably. A variant of this kind of technique is based on tandem hybridization. Tandem
hybridization attempts to compare the annealing of matched versus mismatched probes to
targets (“probe” typically refers to the DNA immobilized on the surface, whereas “target”
generally refers to DNA in solution) over a range of hybridization conditions. Moreover,
tandem hybridization method offers several advantages over traditional oligonucleotide array
configuration, mainly because a unique feature of tandem hybridization is that unlabeled
target DNA is annealed with relatively long-labeled stacking oligonucleotides which bind at a
unique site together with short capture probes positioned immediately adjacent to stacking
oligonucleotides. As a consequence, this system is a highly specific and sensitive one because
capture and stacking probes must be contiguous, in order to obtain a specific hybridization
signal. This system has been successfully applied to RET oncogene and TP53 gene. In this
particular context, we have designed a small microarray directed against the hotspot
mutations that are more commonly observed in exons 5, 7 and 8 of TP53 because in this
region are clustered the most frequent mutations found in clinical samples. The minimum
amount of target detected, as estimated by the proportion of equimolar amount of labeled
stacking oligonucleotide annealed with the sample. It is important to comment that the
intensity of signals seen with the same sample, using 7-mer probes, under traditional, single
tandem and double tandem hybridizations increases from 3 to 6 times in Single Tandem
Hybridization (which uses only a single stacking oligonucleotide) and 6 to 12 times in the
Double Tamdem Hybridization (which uses both stacking oligonucleotides) when compared
to traditional hybridization. Some mutations, such as those causing G’s mismatched, are very
stable, and for this reason are difficult to discriminate with the traditional hybridization
techniques due especially to the length of the probes that are normally used (20-25 nt or even
longer). Short duplexes are considerably more destabilized by even relatively stable
mismatches than longer duplexes, and for this reason short probes (7-mer) have higher
discrimination power. However, short probes when used individually (without tandem
hybridization) are not quite adequate because theses sequences can occur by random chance
multiple times within relatively long targets, which would limit their specificity in the singleprobe
approach. However, this is not the case in the tandem hybridization approach because
the specificity arises from the short probes plus the contiguous stacking oligonucleotides (the
fact that they must be contiguous in order to see a hybridization signal is an important
requirement for this technique). In other words, only 7-mer sites located adjacent to the
stacking oligonucleotides are interrogated, since the stacking hybridization allows only their
detection since isolated 7-mer duplexes (not stabilized by the base stacking) are unstable
under the hybridization conditions used. Finally, in order to decide whether one another
method is adequate to detect point mutations it is important first to know the mutation
frequency expected for any non-selected gene in normal human and in tumor tissue which
makes the analysis less difficult. 

Available Options:
This Item Is Currently Unavailable.
Special Focus Titles
01.Multicultural and Citizenship Awareness through Language: Cross Thematic Practices in Language Pedagogy
02.The Brainstem and Behavior
03.Sustainable Development: The Context of Use of Indigenous Plants for Local Economic Growth
04.Cancer versus Nutraceuticals
05.Fundamentals of Fuel Injection and Emission in Two-Stroke Engines
06.Free to Love: Schema Therapy for Christians
07.Textiles: Advances in Research and Applications
08.Cheese Production, Consumption and Health Benefits
09.Education in Lesotho: Prospects and Challenges
10.Work-Life Balance in the 21st Century: Perspectives, Practices, and Challenges
11.Electrical Measurements: Introduction, Concepts and Applications
12.Potassium Channels in Health and Disease

Nova Science Publishers
© Copyright 2004 - 2018

Mutation Detection in the TP53 Gene: Alternatives in Point Mutation Technologies (pp. 343-345)