Molecular techniques CAQ

🧫 Molecular techniques Created Tags Questions regarding PCR Why taq polymerase can be used on most organisms June 16 , 2025 1210 AM Molecular t echniques 1

Why the nucleotide sequence in RNA primer is important in PCR Comparing PCR and DNA replication Role of primer in PCR Primers are short, single stranded DNA sequences that can anneal to target DNA sequence They are complementary to the regions flanking gene of interest and thus determine the segment to be amplified Molecular t echniques 2

They provide a free 3ʼOH group for chain extensions Moderate temperature by DNA polymers so that the gene of interest can be amplified Role of Taq polymerase in PCR Notes Synthesis complementary DNA strands by adding dNTPs and catalysing formation of phosphodiester bonds between adjacent dNTPs Thermostable DNA polymerase, resistant to denaturation at high temperature Does not need to be added again for each cycle unlike other polymerase and process more efficient Pcr process Molecular t echniques 3

Advantages of PCR It is sensitive as only a minute amount of source DNA is required to amplify a large amount of DNA products By using specific primers, PCR can selectively amplify a particular segment of DNA PCR is fully automated within the thermocycler It can amplify millions of targeted DNA segment rapidly and efficiently Limitations of PCR Taq polymerase lacks proofreading ability as errors occuring early in PCR reaction will get compounded with each replication cycle Success of PCR requires knowledge of sequences flanking target region to be amplified Molecular t echniques 4

DNA fragments to be amplified are limited to about 3kb. Further increases in length of target sequences decrease efficiency of amplification Minute amounts of contaminant DNA may result in unwanted DNA sequences being amplified to significant amounts Why are three temperatures needed during one PCR cycle Heating to Highest temperature 95 degree Celsius) causes hydrogen bonds between complementary bases of each strand to break, causing denaturation of double stranded DNA into single stranded DNA, exposing bases doe complementary base pairing Lowest temperature 55 degree celcius) allows primer to anneal specifically to regions flanking the target DNA sequence via complementary base pairing, where primers determine the segment to be amplified and provide a free 3OH group for chain extensions Moderate temperature of 70 degree celcius is the optimum temperature of Taq polymerase which performs the synthesis of complementary DNA strand Chain extensions occur from 3ʼ end of primer which provides free 3ʼ OH required by polymerase Describe how PCR is carried out to amplify only the region of DNA that is interested Design and use primers with nucleotide sequences Complementary to regions flanking the target gene Ensure that the nucleotide sequence on the primer is not complementary to other regions of DNA After denaturation of DNA double helix, c