"In vitro" Selection of rPrPc-binding RNA Molecules E-Book
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- Herausgeber: GRIN Verlag
- Sprache: Englisch
Internship Report from the year 2014 in the subject Chemistry - Bio-chemistry, grade: 1,0, Free University of Berlin (Institut für Chemie und Biochemie), course: Methodenmodul Nukleinsäuren, language: English, abstract: Aptamers can be considered as nucleic acid based analogues of monoclonar antibodies. These DNA or RNA molecules are characterized by a high affinity and selectivity to the target as well as a huge variety of possible targets, but feature significant advantages compared to antibodies such as the lack of immunogenicity and economic in vitro generation. The preparation of aptamers is based on systematic evolution (see figure 1). Cycles of selection and replication are conducted to narrow down a huge library to aptamers with the best binding properties to the target. In this experiment, an RNA aptamer to the rPrPc protein is selected in vitro. Since a whole SELEX procedures takes several weeks, only one selection cycle is done examplarily.
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Veröffentlichungsjahr: 2014
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Table of Contents
1 Introduction
2 Material and methods
2.1 RNA preparation
2.2 Protein preparation
2.3 In vitro Selection
3 Results
3.1 RNA preparation
3.1.1 T7 transcription
3.1.2 Purification of the transcription product
3.2 Protein purification
3.2.1 Bradford assay of eluted fractions from Ni-NTA
3.2.2 Analysis of protein purification by SDS-PAGE
3.2.3 Analysis of PCR products by PAGE
4 Discussion
4.1 RNA preparation
4.2 Protein preparation
4.3 In vitro selection
1 Introduction
Aptamers can be considered as nucleic acid based analogues of monoclonar antibodies. These DNA or RNA molecules are characterized by a high affinity and selectivity to the target as well as a huge variety of possible targets, but feature significant advantages compared to antibodies such as the lack of immunogenicity and economic in vitro generation.
The preparation of aptamers is based on systematic evolution (see figure 1). Cycles of selection and replication are conducted to narrow down a huge library to aptamers with the best binding properties to the target.
In this experiment, an RNA aptamer to the rPrPc protein is selected in vitro. Since a whole SELEX procedures takes several weeks, only one selection cycle is done examplarily.
Figure 1: Principle of SELEX for RNA aptamers
2 Material and methods
The experiments were conducted following the instructions of the script with minor deviations that are recorded in this minutes.
2.1 RNA preparation
Chemical synthesis of DNA templates was done beforehand by the supervisor. For the T7 transcription, the following pipetting scheme in table 1 was used.
Table 1: Pipetting scheme for T7 transcription (final volume 200 μl)
