steps of c-dna creation and mrna, Biology

C dna is made from a mature m rna of eukaryotic cell with the utilization of enzyme called as reverse transcriptase.

Steps in c dna creation

1. m rna is made and purified from remaining of the rna.

2. The m rna purified is tagged by oligo d t nucleotide as a complementary primer which binds with pol a tail of m rna developing a free 3"oh end that can be extended through reverse transcriptase to develop a complementary dna strand. Now the m rna is eliminated through rnase enzyme leaving a single stranded c dna. This single stranded c dna is changed to double stranded dna with help of dna polymerase.

Diagram in the attachment

The sanger technique, also known as dideoxynucleotide sequencing or chain termination sequencing, is based on the utilization of dideoxynucleotide (ddntp) in addition to normal nucleotides (dntp) found in  c dna. Dideoxynucleotide are basically the same as nucleotides apart from, they include a hydrogen group on the 3' carbon inspite of a hydroxyl group (oh). These modified nucleotides, when incorporated into a cdna sequence, stop the addition of additional nucleotides therefore ends the elongation of the dna chain. This takes place because of phosphodiester bond cannot made between the dideoxynucleotide and the other incoming nucleotide, and therefore the dna chain is terminated.

Steps in sanger technique of cdna sequencing

1.   The area of cdna to be sequenced is amplified and then denatured to create single stranded dna.

2.   A sequencing primer is annealed to form single stranded dna.

3.  Dideoxynucleotide chain termination dna sequencing then gets advantage of the fact that a increasing chain of nucleotides, extending in 5' to 3' direction, will end if, instead of a conventional deoxynucleotide, a 2'3' dideoxynucleotide gets incorporated. By doing four different reactions, each involving a dna polymerase and small amount of one of the four dideoxynucleotides in addition to four deoxynucleotides, four different sets of chain-terminated fragments can be created.

4.  Subsequent to the replication/termination step, these chain terminated fragments will stay bound to the single stranded dna molecule which has acted like a template. Then heating these partially double stranded molecules and adding a denaturing agent like formamide, the single stranded chain termination molecules can be free from their template and separated utilizing high resolution denaturing gel electrophoresis.

5.  The sequence of original region of cdna is then ultimately deduced through examining the associated positions of the dideoxynucleotide chain termination products in the four lanes of the denaturing gel.

Posted Date: 3/6/2013 6:06:50 AM | Location : United States







Related Discussions:- steps of c-dna creation and mrna, Assignment Help, Ask Question on steps of c-dna creation and mrna, Get Answer, Expert's Help, steps of c-dna creation and mrna Discussions

Write discussion on steps of c-dna creation and mrna
Your posts are moderated
Related Questions
Which of the following is true for a toe motor neuron that excites a toe muscle that moves the big toe in the right foot? A. The cell body of the toe motor neuron is located in

Q. Including major events and hormonal changes how can the menstrual cycle be described? One can imagine a cycle like an analog clock at which at 0 o'clock is the beginning and

State about the Complex partial seizures Complex partial seizures, another type of focal seizure, originate most commonly in the temporal lobe. Complex partial seizures are cha

Q. What are the enzymes? What is the significance of enzymes for living beings? Enzymes are proteins that are catalysts of the chemical reactions. From Chemistry it is known as

Q.Explain about Stress Testing in Women? Data on mortality in men is about 2.5 times that of women. At younger ages, however, CAD in men exceeds that found in women by 5 to 1.

How is a genetic trait determined by the genetic code within a DNA molecule? A DNA molecule has 4 dissimilar bases, either CGTA. Any specific combination of these things forms


Fertilization - Development Biology Previously you knew the process which leads to the differentiation of the male and female germ cells, the sperm and ova, respectively. In t

What are universal donors and universal recipients concerning the ABO blood system? Universals donors of the ABO blood type system are the individuals of the type O. Type O blo

A n a e r o b i c lagoon system This specific system of liquid manure handling is restricted to organized dairy farms, where the flushed manure is directed into an anae