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DNA and RNA analyses in detection of genetic predisposition to cancer.

Matyjasik J, Masojc B, Kurzawski G - Hered Cancer Clin Pract (2008)

View Article: PubMed Central - HTML - PubMed

Affiliation: International Hereditary Cancer Centre, Department of Genetics and Pathology, Szczecin, Poland.

ABSTRACT

The introduction of liquid handling robots made possible their application in DNA or RNA isolation, normalization of samples' concentration, PCR preparation, etc. Parallel development of software and hardware enabled complete automatic management of large sample series in genetic testing, including data transfer without any user intervention. Nowadays leading companies offer capillary sequencers analyzing simultaneously up to 96 samples amplified by means of cycling sequencing with fluorescent dyes. Improved "chemistry" and gels' composition enabled accurate sequencing of fragments of 1000 bp in length. Another system (GSFLX machine 2007), based on massive parallel sequencing by cyclic technology, can generate 100 Mb of genomic sequence in a single run (about 4.5 hours). Real-time PCR techniques with TaqMan® probes (or TaqMan® MGB probes) have become commonly used in laboratory practice. MLPA technique used in detection of rearrangements in genes associated with hereditary cancers allows the determination of exon copy number. The presence of deletions or duplications of exons or whole genes can be analyzed by that method. Commercial kits are available for genes with a well-documented association with hereditary cancers: ATM, BRCA1, BRCA2, CHECK1, MLH1, MSH2, MSH6, PMS2, APC, FANCA, FANCD2, PTCH, BMPR1A, SMAD4, TP53, RB1, CDKN2A-CDKN2B, WT1, CDH1, MEN1, NF1, NF2, STK11, SMARCB1.

No MeSH data available.


Principle of RLFP/PCR. View of gel after electroforesis - only variant of gene with G of PCR product will be digests by Cfo I
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Figure 9: Principle of RLFP/PCR. View of gel after electroforesis - only variant of gene with G of PCR product will be digests by Cfo I

Mentions: In the most popular variant of this technique, using agarose electrophoresis, not only flanking primers but also a primer fully complementary to the allele with a mutation or a primer of one which is complementary to the allele with a mutation and another to the wild allele are used. Primers are localized in such a way that different PCR products are of different length depending on the genotype of the examined DNA sample (Figure 9). The modern version of this technique uses short allele-specific probes and machines of real time PCR [22,23]. This allows very fast analysis of many DNA samples. Technology using a template with oligonucleotides immobilized on a solid phase can be considered as a modern version of ASA. A big advantage of this technology is automation and the possibility to analyse even a few thousand known mutations. In many countries the use of such technology is limited due to high costs.


DNA and RNA analyses in detection of genetic predisposition to cancer.

Matyjasik J, Masojc B, Kurzawski G - Hered Cancer Clin Pract (2008)

Principle of RLFP/PCR. View of gel after electroforesis - only variant of gene with G of PCR product will be digests by Cfo I
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2735761&req=5

Figure 9: Principle of RLFP/PCR. View of gel after electroforesis - only variant of gene with G of PCR product will be digests by Cfo I
Mentions: In the most popular variant of this technique, using agarose electrophoresis, not only flanking primers but also a primer fully complementary to the allele with a mutation or a primer of one which is complementary to the allele with a mutation and another to the wild allele are used. Primers are localized in such a way that different PCR products are of different length depending on the genotype of the examined DNA sample (Figure 9). The modern version of this technique uses short allele-specific probes and machines of real time PCR [22,23]. This allows very fast analysis of many DNA samples. Technology using a template with oligonucleotides immobilized on a solid phase can be considered as a modern version of ASA. A big advantage of this technology is automation and the possibility to analyse even a few thousand known mutations. In many countries the use of such technology is limited due to high costs.

View Article: PubMed Central - HTML - PubMed

Affiliation: International Hereditary Cancer Centre, Department of Genetics and Pathology, Szczecin, Poland.

ABSTRACT

The introduction of liquid handling robots made possible their application in DNA or RNA isolation, normalization of samples' concentration, PCR preparation, etc. Parallel development of software and hardware enabled complete automatic management of large sample series in genetic testing, including data transfer without any user intervention. Nowadays leading companies offer capillary sequencers analyzing simultaneously up to 96 samples amplified by means of cycling sequencing with fluorescent dyes. Improved "chemistry" and gels' composition enabled accurate sequencing of fragments of 1000 bp in length. Another system (GSFLX machine 2007), based on massive parallel sequencing by cyclic technology, can generate 100 Mb of genomic sequence in a single run (about 4.5 hours). Real-time PCR techniques with TaqMan® probes (or TaqMan® MGB probes) have become commonly used in laboratory practice. MLPA technique used in detection of rearrangements in genes associated with hereditary cancers allows the determination of exon copy number. The presence of deletions or duplications of exons or whole genes can be analyzed by that method. Commercial kits are available for genes with a well-documented association with hereditary cancers: ATM, BRCA1, BRCA2, CHECK1, MLH1, MSH2, MSH6, PMS2, APC, FANCA, FANCD2, PTCH, BMPR1A, SMAD4, TP53, RB1, CDKN2A-CDKN2B, WT1, CDH1, MEN1, NF1, NF2, STK11, SMARCB1.

No MeSH data available.