What causes transcriptional pausing?

RNA polymerase has an intrinsic capacity for pausing and alterations in any component of the RNA-DNA-RNA polymerase ternary complex can trigger this natural propensity to pause. Specific amino acid residues in Pol II, particularly in the trigger loop domain, are critical for transcription elongation and pausing.

Which end of a PCR primer can be extended by polymerase?

DNA polymerase extends the primer in the 5’→3′ direction. Nucleotides incorporated into the nascent oligonucleotide must be nucleoside triphosphates (i.e. dNTP’s).

When does Taq polymerase stop?

It doesn’t know when to stop, so it keeps synthesizing DNA until the end of the ‘synthesis’ phase of the reaction, when the mixture is heated up to about 95 degrees C, the strands separate, and the Taq enzyme detatches. The mixture now contains long strands of DNA, which are likely to vastly exceed 123 bp in length.

What are the reasons for the reduction in amplification in later cycles of PCR?

The high amount of amplicons in the late cycles of PCR has been proposed to block amplification due to the binding between DNA polymerase and short, blunt-end double-stranded (ds) DNA molecules [7].

What is the function of pause polymerases in cells?

Paused: an early elongation complex that has transiently halted RNA synthesis. Paused polymerase is fully competent to resume elongation, remaining stably engaged and associated with the nascent RNA.

What causes RNA polymerase to pause just after transcription is initiated?

Polymerases that successfully initiate must first break contacts with initiation factors and then interact with elongation factors including the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF). This leads to a reduction of the elongation rate due to an increase in pausing.

How does the polymerase stop in PCR?

It may synthesize hundreds or thousands of extra bases before either: 1) the polymerase just falls of naturally which happens frequently which makes it hard to synthesize very long pieces of DNA by PCR or; 2) the cycle time on the thermocycler ends and the temperature goes up to over 90 degrees and the extra heat …

Why are primers not reusable in PCR?

The primers are not reused — new primers (with the same sequences as before) are needed for each cycle. You need only two types (sequences) of primer, but you need many molecules of each, just as you need many molecules of dATP, dTTP, etc. 7.

Why would PCR amplification eventually slow down and then plateau after around 30 35 cycles?

DNA polymerase after 30-35 cycles is usually denatured, becuse each denaturation temp (95-94) for 30 sec to 1 min effects the protein function and it is not tolerable after 35 cycles usually.

What Causes RNA polymerase II to pause just after transcription is initiated?

Pol II pausing occurs shortly after transcription initiation and involves the association of pausing factors DSIF and NELF. Phosphorylation of DSIF/NELF dissociates NELF from the elongation complex and transforms DSIF into a positive elongation factor that associates with Pol II throughout the gene.