Skip to main content

Recombinant DNA Technology Theory Part

The Recombinant DNA (rDNA) Technology refers to the process of joining foreign DNA segments or DNA segments of interest from different sources and introduced them into a host organism for example a bacterial host. This technology enables the construction of new combination of genes or genetic material to be artificially constructed in the laboratory conditions. These rDNA molecules are introduced into the host cells where they can multiply which is to create, replicate and investigate the recombinant DNA molecules. 

The best and first example of recombinant DNA technology is Human Insulin produced by bacteria, which was first approved by the FDA in 1982. 

A typical Recombinant DNA experiment involves following main steps.

1) Isolation of DNA segments of interest usually gene sequences. (Sequences which are to be manipulated or used and needs to be cloned). 

These selected DNA segments are called as Inserts. 

2) A specific Vector is selected which acts as a CARRIER DNA molecule, which is used to insert the DNA segments (Inserts) by the process of restriction enzyme digestion and ligation. After inserting the DNA molecule into a vector, 
Recombinant DNA = Vector + Insert. 

3) Then these ligated recombinant DNA molecules are transformed into a selected host cell by the process for example Transformation. 

4) After an appropriate amount of time and laboratory conditions, these transformed cells will produce the colonies which may contain the clones (rDNA = Vector+Insert). 

5) Selection of those host cells which carry the rDNA are performed by the process called PCR (Polymerase Chain Reaction) and these clones are used for the library creation or further experiments. 


Each cell in the clone may contain multiple copies of the Insert. 

Image result for recombinant dna technology gif
Image Source: © 2008 Brooks/Cole, Cengage Learning

(The above information is just the overview and main steps involved). 

Practical steps and detailed information will be posted soon. 

Comments

Popular posts from this blog

Nanodrop

Thermofisher, Nanodrop 1000    Nanodrop is a spectrophotometer which is designed to measure the concentration of nucleic acids at 260nm and Proteins at 280nm present in a very small minute volume. It is also capable of measuring the concentrations of dsDNA, RNA, ssRNA and purified protein. Required sample size is usually 1µL. Nanodrop instruments are usually full spectrum spectrophotometer, i.e., Ultra violet and visible range wavelengths (250nm to 750nm).  Nanodrop is commonly used for the quantification of the DNA concentration and RNA concentration in a given sample. However there are many other applications which can be performed on this instrument.  It works on the principle of Spectrophotometer or Sample Retention System....  Principle of Nanodrop: "The Nanodrop works on the principle of sample retention system.. When the small volume of sample is introduced, the arm and pedestal will work together and make the sample in the form of colum...

Statistics in Flow Cytometry Data and "MFI" values

                                 The speed of the flow cytometry offers wide range of data points and data plots. Due to its sensitivity and versatility, it has been used extensively. Flow cytometer can detect up to 1000s of cells per second.. But, it should be noted that the flow cytometry also involves the statistics, its significance, calculating the fluorescence intensity..                Statistics in flow cytometry involves, total number of data points acquired, percentage population, most importantly mean and median fluorescence intensity, and others. Most of us get confused about the term "MFI", whether it means mean fluorescence intensity or median fluorescence intensity.. Truth is, both.. It means it can be described as either mean or median fluorescence intensity. Some researchers use Mean for MFI and some use Median for MFI values.  (Keep...

Compensation in Flow Cytometry (Fluorescence Spill Over Correction).

                      The term "Compensation" in flow cytometers refers to the process of correcting fluorescence spill over. The compensation matrix ensures that the fluorescence emitted is detected in a particular detector which is from the fluorescence being measured. As we all know that most of the fluorescence molecules have broad emission range. if more than one one fluorescence is selected, their emission spectra may overlap. To correct this spectral overlap in cytometry data, a mathematical matrix called compensation is used. The Intention of this article is to teach you the compensation from basic theory to practical. This example for FITC and PE fluorescence molecules shows that, Some of the FITC fluorescence is being detected in PE Detector. and Some of the PE fluorescence is being detected in FITC Detectors. Task here is to make sure that in cytometry data, FITC is in FITC detector only and PE is in PE Detector only....