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Prof. Allen Gathman has a great 10-minutes video on Youtube, explaining the reaction of adding nucleotide in the 5' to 3' direction, and why it doesn't work the other way. Briefly, the energy for the formation of the phosphodiester bond comes from the dNTP, which has to be added. dNTP is a nucleotide which has two additional phosphates attached to its 5' ...


10

DNA replications needs a source of energy to proceed, this energy is gained by cleaving the 5'-triphosphate of the nucleotide that is added to the existing DNA chain. Any alternative polymerase mechanism needs to account for the source of the energy required for adding a nucleotide. The simplest way one can imagine to perform reverse 3'-5' polymerization ...


9

Short Answer In a nutshell, DNA sequencing technology has a limit to how long a stretch of DNA it can read in one go. Long Answer So what most commonly occurs is the length of DNA you wish to sequence needs to be (almost randomly) chopped up into given lengths (depending on the technology) and each length or read is sequenced in parallel. But now you ...


7

The RNA world hypothesis states that self-replicating RNA (that is, an autocatalytic RNA polymerase) was the first form or precursor of life. So, in that context, your question is basically asking how life originated. The obvious answer is that we don't know (currently anyways), but I'm going to take this opportunity to describe a few really neat experiments ...


7

Unlike in an SDS-PAGE, where the SDS adds negative charges to all proteins, in a standard agarose gel for nucleic acids all proteins should move according to their own charge. You can get a rough idea in which direction Taq would move by getting the amino acid sequence and calculating the isoelectric point at the pH you are running your get at. It might be ...


4

According to their website New England Biolabs use a version of the approach pioneered by Wayne Barnes, as described in: Kermekchiev, M.B., Tzekov, A and Barnes, W.M. (2003) Nucl. Acids Res. 31, 6139–6147 This is basically an assay for the mutation rate in a PCR-amplified lacZ (β-galactosidase) gene, assayed by transforming E. coli, plating on the ...


3

Transcription always proceeds in the direction 5' (5-prime) to 3' (3-prime) on the coding strand of DNA. Binding of both transcription factors and RNA polymerase to DNA depends on sequence motifs in the DNA. Transcription always happens in the same direction with respect to the chemical structure of the coding DNA strand, while the transcription direction ...


3

Deep sequencing is naturally error prone. Sequencing will never be perfect, because no enzyme will ever perform 100.00000% perfectly. In Illumina sequencing, you put your starting molecule down on the flowcell, then the polymerase makes a cluster of copies around it. But at each step of building each copy, there's a chance the polymerase will make a ...


3

What makes you think that the lac repressor will still be bound at the target gene? In strains with this type of regulatory configuration (usually with DE3 in the genotype) the idea is that IPTG induction turns on expression of T7 polymerase from a lac promoter on the chromosome, and then this T7 polymerase then transcribes the target gene on the plasmid. ...


2

Actually there is a polymerase that catalyzes 3' - 5' elongation. See for example the Thg1 superfamily. "Doing it in reverse: 3'-to-5' polymerization by the Thg1 superfamily." Jackman, et al.


1

Another important point is that we almost always sequence a population of cells, not just a single cell. Cancer cell populations, for example, can have multiple sub-populations of cells with different genetics (subclones). Deep read sequencing allows us to see this heterogeneity, and also measure the abundance of each sub-clone. Even with a single cell, we ...


1

I think that you have a couple of points wrong. Since your question is asked using bacterial terminology, I'll stick to that. The leading strand, the one that is initiated at the origin of replication, is synthesised by pol III which is a highly processive polymerase, i.e. it keeps on going for long periods, making very long products. In principle a single ...


1

You can look up Gibson Assembly or Circular Polymerase Extension cloning (CPEC). For both of these the website for J5 has some good protocols. Here is the one for CPEC: http://j5.jbei.org/j5manual/pages/80.html For CPEC you can look at the 2011 Quan paper: http://www.ncbi.nlm.nih.gov/pubmed/21293463 Hopefully that helps.


1

it is highly variable even in a single cell type. Dont know for a human cell but this is the range for ecoli: 1500-11400 check this site out. got this number from there (its quite useful for questions like these): http://bionumbers.hms.harvard.edu/bionumber.aspx?&id=101440&ver=3&trm=RNA%20polymerase You can estimate protein copy numbers and ...


1

In my opinion, Prof. Allen Gathman's "great 10-minutes video on Youtube", is a pretty waste of time, if you already know how hydrolysis happens. In fact, he has not considered the 3'->5' route in an unbiased approach; he doesn't seem to look at the possibility of triphosphate appearing on the growing strand (in the video). Actually, the only difference ...



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