TAILIEUCHUNG - analysis of genes and genomes phần 5

Hình . RT-PCR. Các Enzyme Transcriptase Đảo ngược sử dụng các oligonucleotide bổ sung cho sự tổng hợp DNA chính từ một phân tử RNA. Những sợi DNA đơn được sản xuất Sau đó là sử dụng như một khuôn mẫu cho sự tổng hợp của sợi DNA thứ hai, | 178 POLYMERASE CHAIN REACTION 4 5 mRNA AAA-3 3 5 Primer 1 RT 5 3 mRNA AAA-3 5 DNA Primer 2 5 3 3 5 Taq ị 5 3 3 5 Primer 1 3 5 3 5 3 5 3 5 Primer 2 Amplified product Figure . RT-PCR. The enzyme reverse transcriptase uses a complementary oligonucleotide to prime DNA synthesis from an RNA molecule. The single strand of DNA produced is then used as a template for the synthesis of a second DNA strand and then for amplification by PCR devised as a method of RNA amplification and quantitation after its conversion to DNA. RT-PCR can be used for cloning cDNA library construction and probe synthesis. The technique consists of two parts Figure - the synthesis of DNA from RNA by reverse transcription RT and the subsequent amplification of a specific DNA molecule by polymerase chain reaction PCR . The RT reaction uses an RNA template typically either total RNA or polyA RNA a primer random or oligo dT primers dNTPs buffer and a reverse transcriptase enzyme which we will discuss more in Chapter 5 to generate a single-stranded DNA molecule complementary to the RNA cDNA . The cDNA then serves as a template in the PCR reaction. During the first cycle of REAL-TIME PCR 179 PCR the single DNA strand is made double stranded through the binding of another complementary primer and the action of Taq DNA polymerase. Like other methods of mRNA analysis such as northern blots and nuclease protection assays RT-PCR can be used to quantify the amount of mRNA that was contained in the original sample. This type of analysis is particularly important for monitoring changes in gene expression. However because PCR amplification is exponential small sample-to-sample concentration and loading differences are amplified as well. Even large differences in target concentration 100-fold or more may produce the same intensity of band after 25 or 30 PCR cycles. Therefore RT-PCR requires careful optimization when used for quantitative mRNA analysis. Quantitation usually takes one of two forms - .

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