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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5539_Библиотеки_им_академика_М_И_Перельмана.pdf
Mpoke S
Kenya Medical Research Institute, Center for Biotechnology Research and Development,
Kenya
Mbuchi M
Kenya Medical Research Institute, Center for Clinical Research, kenya
Muthami L
Kenya Medical Research Institute, Center for Public Health and Research, Nairobi,
Kenya
Mathaai R
Department of Biochemistry, University of Nairobi, G.P.O, Nairobi, Kenya
Otieno P
Kenya Medical Research Institute, Center for Biotechnology Research and Development,
Kenya
Nyakundi P
Kenya Medical Research Institute, Center for Biotechnology Research and Development,
Kenya
Johannes Routila
Centre for Biotechnology, University of Turku, Turku, Finland
Jukka Westermarck
Centre for Biotechnology, University of Turku, Turku, Finland
Department of Pathology, University of Turku, Turku, Finland
Davide Onofrio Fontana
Area di Geriatria, Policlinico Universitario Campus Bio-Medico, Roma, Italy
Claudio Pedone
Area di Geriatria, Policlinico Universitario Campus Bio-Medico, Roma, Italy
Fondazione “Alberto Sordi”, Roma, Italy
Raffaele Antonelli Incalzi
Area di Geriatria, Policlinico Universitario Campus Bio-Medico, Roma, Italy
Fondazione “Alberto Sordi”, Roma, Italy
Fondazione “San Raffaele - Cittadella della Carità”, Taranto, Italy
Nadeem Kizilbash
Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences,
Northern Border University, Arar-91431, Saudi Arabia
xix
Majed Alrowaili
Department of Surgery, Faculty of Medicine, Northern Border University, Saudi Arabia
xx
PREFACE
The human genome study and genetics have emerged as a key concept and
important aspect of public health and medicines. The complete characterization
of genetic disorders and other related diseases can offer valuable information
regarding the diagnosis of the patients. Moreover, the familial mutation
identification, may lead to proper genetic counseling within families that may
prevent genetic disorders in future pregnancies through possible preimplantation
genetic diagnosis or prenatal diagnosis. In recent years the area of clinical
molecular diagnostics has evolved considerably with the advances in fundamental
research and technologies of human genetics. Earlier, the researchers used to
depend on preliminary techniques to assess genetic mutations.
Over the years more and more assay techniques were implemented for better
utilization of clinical molecular diagnostic. Previously, the assays were usually
targeted to more common disorders like cystic brosis and hemoglobinopathies
using methods linked indirectly to mutation detection by linkage and haplotype
analyses. These processes were very laborious, requiring a large amount of
DNA from the patients and extensive knowledge of the region of the genome
in question. Even then the results acquired were not very easily interpretable.
However, those earlier analysis methods given the much-needed foundation for
the molecular diagnostics and several of such techniques are still in use.
The molecular diagnostic techniques got revolutionized with the advent of PCR
or polymerase chain reaction which was first introduced by Mullis et al., 1986.
It has the capability to produce numerous copies of any region of the target DNA
enabling faster analysis as well as direct identification of mutations. In no time
the earlier assays were modified to integrate the use of DNA amplified using
PCR instead of the genomic DNA. Gradually, the assays dependent on allelespecific detection are developed into systems with high-throughput allowing
larger scale analysis of patient samples. The discovery of PCR also enabled
laboratories to identify rare disorders, along with the most common ones.
In this regard, the NGS or next-generation sequencing methods are fetching
more and more attention. But, the most common method for analysing many
genetic disorders seems to be automated Sanger sequence analysis and is used
in most of the molecular diagnostic laboratories for clinical purposes.
The choice of assay, however, depends largely on the alleles or gene of interest
and number of patients needed to be screened.
In this Book, an attempt is made to offer an insight on the common molecular
techniques that are in use and recent advances in molecular diagnostics.
Editor
xxii
INTRODUCTION
Preethi Kartan
Types of molecular diagnostic techniques developed to
improve the medical diagnosis process
TARGETED ANALYSES
Allele-specific methods to detect mutation were the first analysis
technique developed for implementation in clinical diagnosis. It was
developed in the early 1980s and is still used regularly in clinical
laboratories because of its ease of use and means of simple conversion
for high-throughput applications. The drawback is that this technique can
be used only to diagnose known mutations or polymorphisms. However,
combining additional assays full comprehensive detection of mutations
is possible.
Advances in Molecular Diagnostics
2
RESTRICTION FRAGMENT LENGTH
POLYMORPHISM (RFLP)
This is one of the oldest techniques used in molecular diagnostics for
clinical purpose. In this method changes the genome were detected through
Southern blotting followed by RFLP. This method was developed in 1975
(Southern, 1975) and disorders like cystic brosis, phenylketonuria and
thalassemias were among the diseases first described using RFLP. It was
nevertheless a laborious process.
Allele-Specific Hybridization of Oligonucleotides
Allele-specific oligonucleotide (ASO) hybridization is also known as dot
blot analysis. This was an early attempt to identify specific mutations
responsible for a particular disorder. Here to help of radio-labelled probe
is taken to identify mutant alleles. It was used extensively in the early
1980s to detect diseases such as sickle-cell allele and β-thalassemia in
parents (Conner et al., 1983; Orkin, Markham, & Kazazian Jr, 1983).
Amplification Refractory Mutation System (ARMS)
ARMS is a PCR-based technique developed in the later part of the 1980s
which was used to analyze point mutations. It identifies the difference
between the DNA template and the 3′ end of a PCR primer (Newton
et al., 1989). Thus, simple amplification by PCR can differentiate the
alleles and multiple primer sets may be used at the same time in one
tube to analyze multiple mutations simultaneously. Disorders like cystic
fibrosis, mitochondrial mutations, and phenylketonuria can be identified
by ARMS (Venegas & Halberg, 2012).
Pyrosequencing
This technique is based on DNA sequencing technique utilizing real-time
DNA synthesis, detection by luminescence monitoring. It was described
first in 1985 while monitoring the DNA polymerase activity continuously
using enzymatic methods (Nyrén & Lundin, 1985). This technique was
later modified to enhance the analysis of genetic variations such as known
mutation detection or SNP genotyping.
Introduction
3
Real-Time PCR
Here the DNA or RNA quantification and analysis is done while they
are developed in real-time with the help of the TaqMan probe or DNA
intercalating fluorescent dyes and no post-PCR manipulation is made.
This sensitive assay technique got popular in the middle of 1990s
(Gibson, Heid, & Williams, 1996).
This technique is used to detect nucleic acids rapidly for diagnostic
purpose. It helps fast and accurate detection of several infectious diseases,
genetic abnormalities and cancer. Emerging infectious diseases such as
newly discovered strains of u can be effectively detected using Real-
Time PCR (Espy et al., 2006; Huggett, Cowen, & Foy, 2015).
Figure: Real-time PCR method (Deininger et al., 2009).
UNKNOWN MUTATION DETECTIONS
The techniques mentioned thus far need to have a prior knowledge on
the mutation to be analyzed and the sequence of nucleotides around it. To
cover unknown mutations in the targeted region the following molecular
diagnostic techniques can be used:
• Gradient Gel Electrophoresis (GGE): GGE technique
involves denature (DGGE) and temperature (TGGE) based
principles utilizing the alteration of electrophoretic mobility
of all the fragments of double-stranded DNA by partial
Advances in Molecular Diagnostics
4
denaturation. This method was rst used in 1985 to characterize
human β-thalassemia mutations (Myers, Lumelsky, Lerman,
& Maniatis, 1985) allowing identify allelic changes without
having the knowledge of the exact DNA sequence that region.
Simultaneous screening of multiple nucleotide changes is also
possible.
• Heteroduplex Analyses (HDA) and Single-Strand
Conformation Polymorphism (SSCP): HDA and SSCP are
the two techniques developed closely after the introduction
of PCR (Orita, et al., 1989) which utilizes the fact that in a
non-denaturing gel, short single-stranded fragments of DNA
fragments migrate as a function of size and their sequences. It
has the advantages of introducing non-radioactive labeling of
the PCR products, lower cost, and greater reproducibility.
• Denaturing High-Performance Liquid Chromatography:
This is another method rst available in 1997 (Underhill et
al., 1997) which combined all the best available techniques of
that period. However, it is a labor intensive process requiring
numerous analysis and optimization by gel electrophoresis. It
enabled a large number of sample analysis in a high-throughput
facility, but could not differentiate between different mutations
present in the same fragment.
• ProteinTruncationTest(PTT): PTT test or in vitro synthesized
protein assay is not relying on genomic level changes but on
analysis focusing on the size changes of the proteins due to
in vitro translation and transcription of the target gene (Den
Dunnen & Van Ommen, 1999). Thus the test is not a very
common method used during the screening of mutations in
most genes, but can effectively determine specic gene targets.
• Sanger Sequencing: Extensive designing an optimization is
required to make the above methods sensitive even though
they are easy to perform and Sanger sequencing is again
required for conrmation. Therefore, Sanger sequencing with
capillary electrophoresis is usually performed nowadays as a
molecular diagnostic tool. The Sanger method of sequencing
was, however discovered in 1977 (Sanger, Nicklen, & Coulson,
1977).
Introduction
5
Next Generation Sequencing (NGS)
NGS or high-throughput sequencing utilizes many different advanced
sequencing techniques such as Illumina sequencing for molecular
diagnosis. This technique has revolutionized the process of characterizing
cancers at the epigenetic, transcriptomic and genetic levels. It also
allowed cataloging of every mutation, somatic rearrangements and
copy number aberrations in a whole cancer genome at the level of base
pair resolution. It is also possible to carry out transcriptomic analysis
of noncoding RNAs, small RNAs and mRNAs as well as other assays
including high-throughput chromatin immunoprecipitation and genomewide methylation assays (Reis-Filho, 2009).
Copy Number Variations
In the diagnosis of many diseases such as congenital anomalies,
developmental delay, and intellectual disability the analysis of
chromosomes is essential. Routine analysis of chromosomes can detect
copy number variations including structural rearrangements (balanced
and unbalanced) or duplications (greater than ~5 Mb) and aneuploidy of
the whole chromosome.
Southern Blotting
Along with RFLP, this is a common method employed to identify
mutations for any specific disorder. It is used extensively in PCR-based
analysis, providing additional information on diseases such as fragile X.
Tandem PCR analysis is also performed.
Multiplex Ligation-Dependent Probe Amplification
(MLPA)
MLPA is a very effective assay technique to detect variations in copy
number as it can analyze multiple regions at the same time at less cost.
The process requires a PCR thermocycler with capillary electrophoresis.
It is more of a combination of AFLP and MAPH (Schouten et al., 2002).
Here simultaneous amplification of multiple oligonucleotides in a single
set is possible without immobilizing the sample to a membrane. No need
to remove excess probe.
Advances in Molecular Diagnostics
6
Array Comparative Genomic Hybridization
This is another technique developed in the early 1990s (Schouten et al.,
2002) which are used widely to identify and characterize chromosomal
abnormalities in various types of cells. It can detect deletions or
duplication of even a small single exon (Landsverk, Wang, Schmitt,
Pursley, & Wong, 2011).
Single-Nucleotide Polymorphism (SNP) Array
SNP arrays were designed at first for human DNA genotyping using
simultaneous analysis of over thousands of SNPs in various regions of
the genome (Wang et al., 1998). It is now widely used to detect changes
in copy number and heterozygosity absence.
Immunoassays
Producing inflammatory response with the help of chemical factors
such as cytokines (specialized chemical mediators) which correspond to
systemic conditions in patients is a useful tool. This is done by initiating
different infections or process of inflammatory processes deliberately
to assess the immune response in patients. In cardiac patients high
interferon-beta level in serum corresponds to spontaneous enterovirus
elimination from myocardial tissue which reduces the mortality rate
in cardiac patients (Uwe Kühl, Lassner, von Schlippenbach, Poller, &
Schultheiss, 2012). Immunoassays such as indirect immunoassay, line
immunoassay and radioimmuno-precipitation are widely used for the
diagnosis of HIV (Arya, Lal, Singh, & Kumar, 2015).
Endomyocardial Biopsy
This tool is used to survey cardiac allograft rejection as well as to evaluate
the dilated and restrictive cardiomyopathies (lesser extent). It is an
essential technique, particularly applied when other diagnostic methods
fail to produce a proper diagnosis. For a conclusive tissue diagnosis,
optically controlled myocardium must be made during the sectioning
which allows for having a myocardium proportion exceeding 80% which
can be evaluated. Moreover, a complete analysis of the tissue samples is
necessary for accurate diagnosis.