Question

1) What happens to DNA synthesis when a dideoxyribonucleoside triphosphate ( ddNTP) is added to a growing DNA chain ( rather than the normal deoxyribonucleoside triphosphate) ?

2) About how many copies of DNA are made after 10 cycle of PCR ?

3) What are we doing when we amplify a particular DNA segment ?

4) How are DNA fragments stored in a cDNA library ?

5) What is the basic purpose of a DNA microarray ?

Please answer all questions . Thanks

Answer 1

1)If a ddNTP is added to a growing DNA chain,since it lacks a hydroxyl group at the 3' carbon of the sugar moiety,which is incorporated to the end of the dna chain,DNA synthesis stops because a phosphodiester bond cannot be formed with the next incoming nucleotide.This is the principle of termination of dna synthesis.Instead of adding dNTP,ddNTP's are added to stop the process.They are also called as chain inhibitors of dna polymerase.

2)The number of double stranded DNA pieces doubled in each cycle,so after n cycles there will be 2^n copies of dna.ie here there are 10 cycles,so the number of dna copies after 10 cycles is 2^10 = 1024 copies.A single molecule of DNA can be amplified thousand,millions and billions of time.The technique used to amplify dna is PCR,polymerase chain reaction.

3)During amplification of a DNA segment,the sample is first heated so the DNA denatures,or seperates into two pieces of single stranded DNA.Then adding an enzyme called taq polymerase synthezises or builds two new strands of DNA using the original strands as templates.This results in the duplication of the original DNA,with each of the new molecules containing one old and one new strand of DNA.Then each of these strands can be used to create two new copies.The cycle of denaturing and synthezing new DNA is repeated as many as 30 or 40 times,leading to more than one billion copies of the exact DNA segment.

4)A cDNA library is a comibination of cloned cDNA fragments inserted into a collection of host cells,which constitute some portion of the transcriptome of the organism and later they are stored as library.If mRNA is purified,oligo-dT is tagged as a complementary primer which binds to the poly-A tail providing free 3'-OH end which is extended by reverse transcriptase and creates complemntary DNA.mRNA is removed by rnase and single stranded complementary dna is converted to double stranded dna .In a cDNA library there it stores a collection of genes that are encoded into proteins.

5)A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time.They are microscopic slides that are printed with thousands of tiny spots in defined positions,with each spot containing a known dna sequence or gene.It is mainly used to determine whether the dna fom a particular individual contains a mutation in genes.This technology is mainly based on differential hybridization strategy.