Reverse Transcription Polymerase Chain Reaction Lab Report

1.INTRODUCTION

The biological science field has been progressing in such a brisk motion ever since it was introduced to the world. Renowned scientists and inspiring researchers continuously looking for the loopholes and rooms for improvements for the betterment of today’s society. And all this is circling on the foundation of the field itself that is gene. Gene is a part, a tiny piece of genetic material in a code and world-widely known as DNA. In other words, it is the functional unit of heredity and acts as instruction to make molecules in later process which is protein that dictates cell function. Every person has two copies of each gene - inherited from each parent and they are located on chromosomes.

The genes cannot be used unless they
It is then separated and sorted in terms of size by gel electrophoresis. Afterwards, it is transferred onto a membrane called blotting. It is baked at 80ºC before labeling of DNA probe for hybridization takes place. The sequence of the probe is made complementary to a specific RNA sequence in the sample. Moreover, a radioactive atom or a fluorescent dye is used as label on probe.

Reverse Transcription Polymerase Chain Reaction (RT)-PCR
Employing PCR-based procedure in analyzing RNA would require cDNA to be synthesized using reverse transcription. When reverse transcription and polymerase chain reaction are combined together, it will permit detection of low abundance RNAs. RT-PCR is distinct from Real Time Polymerase Chain Reaction (qPCR). The latter measures the amplification of DNA in quantitative way and it uses fluorescent dyes.

The process begins by heating the cDNA at 95ºC to denature it. Thereof, the hydrogen bonds between complementary strands are disrupted. It then produces single-stranded molecules. In subsequent step, annealing of primers that are complementary to the target sequence is realized by lowering the temperature. The final step is extension in which DNA synthesis happens. Temperature of choice in the last step is usually 72ºC and it allows the synthesis of a new strand that complements to the
Revelation of the 3′-most restriction site anchored to the oligo(dT) bead is done after synthesized cDNA on oligo(dT) is digested together with the anchoring enzyme NlaIII (AE). Then, different linkers are put into use in the ligation. Ditags are yielded when SAGE tags liberated from the oligo(dT) beads undergo separation, blunting and ligation. Last but not least, they are amplified using PCR, liberated from the linkers, purification of gel, serially ligated, cloned, and sequenced using an automated