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Biolad:
QUOTE: SoftRat,
You said:
“If this is true Bio Lad then why were you not up front about the fact that cDNA is made via template switching? Why did you clearly imply that I was off base on this?�?BR>
I “clearly implied that you were off base�?because you were. I will explain more in depth below.
“Biolad you are hiding relevant information here. As a molecular biologist you should not have to do that if Hep C virus really does exist.�?BR>
Misunderstanding of the material discussed on your part in no way indicates a “hiding�?of data on my part. I would again stress that calling people frauds or liars (as you insinuated of me by accusing me of hiding data) is neither conducive to a debate nor is it called for, especially when it is, again, due to your misunderstanding of data. The reason I wanted to wait to post this was to get it all in one post as Rod had previously told me to keep the number of fragmented posts to a minimum.
To your first question then:
“why were you not up front about the fact that cDNA is made via template switching�?BR>
Your misunderstanding here seems to stem from the lack of distinction between “template switching can occur�?and “template switching always occurs�?
A cDNA is a DNA copy of an RNA template. Template switching under the controlled environment of an experimental setup does not happen as often as you seem to think. When it does occur, it is between areas of high homology between the original template and the new template (this will be a very important factor later on). Very few cDNAs are the result of such an event under controlled experimental conditions unless the experiment is designed specifically to test this (such as the Luo and Taylor paper). So how can we be sure that the HepC genome is not the result of one of these events? I will go through, step by step, how I would verify this. Please read it all the way through before trying to come up with objections as the points will build on each other.
The way in which the complete HepC genome was sequenced refutes the idea that it was the result of template switching. (This is relevant to the Chiron paper isolation because of the sequence match the with the portion they isolated) Briefly the following was done:
First:
Serum was isolated and spun down in a centrifuge. The viral fraction (the fraction at which lower weight particles would be contained) was isolated and spun down again and the RNA extracted from the pellet. This fraction would contain no cells as a source of RNA as those would be in a different fraction.
Next:
The RNA was reverse transcribed using random 6-base primers (this is important as we will see shortly) creating a pool of cDNA copies of various lengths (dependant on which two random primers created the fragment) which were inserted to Lambda phage vectors.
Then:
A screening is done to see if any peptide sequences expressed will react to enzyme linked assays. This is NOT however a step taken to isolate which clones they will chose for sequencing. This is evident by the fact that only 9 clones chosen displayed EIA reaction when many more are used in the construction of the final sequence.
The sequences were hybridized to human placental DNA to remove any clones that may have been of human genomic origin. This is also important for the following reason. IF the HepC genome was nothing more than a collection of Template-Switched human RNA then the homologous regions needed to initiate said switching would hybridize with the human DNA. In fact, if all or even many of the segments of cDNA that they isolated were the result of template switching (as you claimed) then MANY homologies would be present, even if the HepC sequences were supposedly in various parts of the genome. Yet, no homologies were found to the coding regions (Table 1). This again refutes the Template-Switching origin of HepC idea.
Lastly;
Clones are then sequenced and placed in order based on overlapping sequences. This organization of the sequences is similar to how the human and chimp genomes were put together. Also of importance is that the overlaps match to nucleotide identities of more that 99.3% (page 2). If there were template switching, this would not be the case.
Summary:
1) Template Switching can occur but does not necessarily HAVE to.
2) When it does occur it is between areas of homology.
3) Thus, if HepC were the result of Template Switching, it would have incorporated cellular RNA and would show homology to the human genome.
4) If the ORFs for HepC proteins are interspersed throughout the genome there will be more possible areas of hybridization and thus more identified areas of homology.
5) There are no such areas of homology found in the hybridizations performed.
6) There is likewise >99.3% nucleotide match between overlapping cDNA clones during the sequencing. This would not be the case if the clones were the result of random chimeric RNAs.
7) Thus, the HepC cDNA isolates are not the result of Template Switching. And, since the Chiron sequence matches the HepC genome sequence, it too is not a result of Template Switching.
8) The lack of homology to human chromosomal DNA also shows that this is not of human genomic origin.
9) Thus, the only conclusion one can come to based on this data is that HepC is of exogenous origin and (by the continuous overlapping sequences) of a single molecule.
One last the idea of template switching and interspersed HCV genome.
Now if, as you claim, the genome required to code the proteins found in HCV is interspersed throughout the human genome and the ~10kb genome sequenced by virologists is all a fraud, then one would not expect to be able to directly detect the ~10kb RNA sequence in infected human serum, right? Unfortunately this has been done. The following paper cited at the end of this section carries out a Northern Blot analysis of serum from infected and uninfected individuals using the conserved 5’UTR probe. They found the band corresponding to the ~10kb marker only in the infected individual. This additionally refutes your claim that the HCV genome is nothing more than an artifact of RT-PCR as no reverse transcription ever took place.
References:
Genome Sequencing paper available in full here (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=239876&blobtype=pdf)
“Direct Detection of Circulating Hepatitis C Virus RNA Using Probes from the 5' Untranslated Region�?available full text here (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=295919&blobtype=pdf)
So how many cDNAs really are the result of Template Switching?
Back in college my thesis work focused on Arabidopsis thaliana (a plant whose genome was fully sequenced by that time) and I had to use RT to make a cDNA copy of a gene for an expression vector. I probably still have the old sequence files somewhere of the cDNA clones I obtained but suffice it to say that there were no chimeric sequences. Don’t take my word for it, though, because you can check for yourself! Try the following:
Look a gene in an EST database. ESTs are cDNA clones generated by RT and inserted into cloning vectors (much the same way they sequenced the HepC genome, but different vector). For ease of this exercise use human or chimp genes as the whole genome is already sequenced and relevant to this discussion. Got a gene? Now compare it to its genomic counterpart via BLAST and compare the two. Other than the removed introns, the coding regions should match up. You can repeat this as often as you wish.
About sequencing the RNA directly:
You have stated at least twice now that to prove that HepC exists that the RNA must be directly sequenced. As I have told you before that is technically more difficult to do and I do not know of anyone who does this anymore as it requires the use of radiolabeled ribonucleotides.
-BioLad UNQUOTE.
Softrat:
QUOTE: “This fraction would contain no cells as a source of RNA as those would<o:p></o:p>
be in a different fraction.�?lt;o:p></o:p>
<o:p></o:p>
It is well known that the mRNAs called Hep C are found in high titers outside the cells, in the blood. This is the case even though no matching levels of particles are found. Dennin commented on this. This is not consistent with the existence of a virus. But it is consistent with the existence of cellular mRNA being produced in large amounts under toxic stress.<o:p></o:p>
<o:p></o:p>
“No convincing support has been provided so far for the existence of extrahepatic hepatitis C virus particles that should correspond to the sometimes extremely high concentration of 'HCV-RNA' in serum or plasma.�? Dennin
<o:p></o:p>
Bio lad said
<o:p></o:p>
“…creating a pool of cDNA copies of various lengths�?lt;o:p></o:p>
<o:p></o:p>
Earlier you told me there was only one cDNA. I told you there were many and you lied about this and said there was only one. This pool of cDNA is exactly what Dr. Lanka was talking about. <o:p></o:p>
<o:p></o:p>
“The resulting pieces of DNA too, are necessarily both shorter and longer than the "correct" length…�?-Dr. Lanka
<o:p></o:p>
This shows you are hiding relevant information and now you are revealing it cause I have shown that template switching is involved in making cDNA.
<o:p></o:p>
Also those cDNA of various lengths exist precisely cause template switching is going on as Dr. Lanka, a PhD molecular biologist, has explained. <o:p></o:p>
<o:p></o:p>
“Your misunderstanding here seems to stem from the lack of distinction<o:p></o:p>
between "template switching can occur" and "template switching always<o:p></o:p>
occurs".�?�?Bio Lad<o:p></o:p>
<o:p></o:p>
Well it is occurring here Bio lad. The different lengths of cDNA prove that. Some strings used more templates and some used less depending on which random primers were involved. Just like Dr. Lanka said.<o:p></o:p>
<o:p></o:p>
<o:p></o:p>
“IF the HepC genome was nothing more than a collection of Template-Switched human RNA then the homologous regions needed to initiate said switching would hybridize with the human DNA.�?lt;o:p></o:p>
<o:p></o:p>
Oh but Biolad homologous regions are not needed to initiate switching. You have been caught in a big big lie.<o:p></o:p>
<o:p></o:p>
<o:p></o:p>
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=334354 <o:p></o:p>
<o:p></o:p>
<o:p></o:p>
<o:p></o:p>
“Temperature-dependent template switching during in vitro cDNA synthesis by the AMV-reverse transcriptase.�?lt;o:p></o:p>
<o:p></o:p>
“During in vitro cDNA synthesis, the avian myeloblastosis virus (AMV) reverse transcriptase can switch from one template to another in a homology-dependent and temperature-dependent manner.�?BR>
<o:p></o:p>
So you see during cDNA manufacture homology is just one way to do template switching. Temperature dependent template switching also exists. I wonder how many other ways there are to do template switching? But Bio Lad, it is clear I cannot rely on you to tell me all the ways template switching is done. Once again Bio Lad you have been caught red handed hiding very important information. You cannot be trusted. If Hep C and other viruses did exist they you would not need to hide information like this.
<o:p></o:p>
So template switching can be made to happen with out homologous regions. Thus Hep C can be made from template switched human mRNA. And this fake Hep C cDNA would not hybridize at all. Cause there would be no homologous regions to do this.
<o:p></o:p>
“In fact, if all or even many of the segments of cDNA that they isolated were the result of template switching (as you claimed) then MANY homologies would be present, even if the HepC sequences were supposedly in various parts of the genome. Yet, no homologies were found to the coding regions (Table 1). This again refutes the Template-Switching origin of HepC idea.<o:p></o:p>
<o:p></o:p>
No Biolad it shows that template switching was done that did not involve homologies.
Again:
<o:p></o:p>
“During in vitro cDNA synthesis, the avian myeloblastosis virus (AMV) reverse transcriptase can switch from one template to another in a homology-dependent and temperature-dependent manner.�?BR>
<o:p></o:p>
This shows that some reverse transcription enzymes can function either way. This would explain why some cDNA was removed when:
<o:p></o:p>
“The sequences were hybridized to human placental DNA to remove any<o:p></o:p>
clones that may have been of human genomic origin.�?lt;o:p></o:p>
<o:p></o:p>
And of coarse there may be other reverse transcription enzymes that template switch based on other variables you have not told me about. You do like to hide the data to deceive people Bio Lad.<o:p></o:p>
<o:p></o:p>
I just love Bio Lad how you told me there was only one cDNA and now you have been forced to admit that there was all kinds of crap in this stew of many little mRNAs. This is exactly what I was talking about. And now you have been forced to admit it cause I showed that cDNA is made via template switching.<o:p></o:p>
<o:p></o:p>
Bio lad said:
<o:p></o:p>
“Clones are then sequenced and placed in order based on overlapping<o:p></o:p>
sequences. This organization of the sequences is similar to how the<o:p></o:p>
human and chimp genomes were put together. Also of importance is that<o:p></o:p>
the overlaps match to nucleotide identities of more that 99.3% (page<o:p></o:p>
2). If there were template switching, this would not be the case.�?lt;o:p></o:p>
<o:p></o:p>
For this I need the aid of virologist Dr. Stephan Lanka again. Let see what the good doctor has to say.<o:p></o:p>
<o:p></o:p>
“The resulting pieces of DNA too, are necessarily both shorter and longer than the "correct" length of HIV. Pieces corresponding to the "correct" length of HIV must be selected for size, because otherwise the purported DNA preparation would be a mixture of various lengths, which would violate a cardinal rule of virology that all nucleic acid of a particular virus be identical in size. �?BR>
“Having artificially prepared DNA pieces of uniform length, they are still not ready for presentation, because they consist of a mixture of all kinds of RNA fragments transcribed into DNA and thus cannot be shown to represent unique viral DNA. Accordingly, the mixture is subjected to a kind of lock-and-key detection process called hybridisation, whereby pieces of DNA are detected which complement more or less a probe of that which it is desired to be shown to have been prepared. �?BR>
“Since no DNA from HIV existed to hybridise with the prepared DNA, Gallo and Montagnier simply used stretches of DNA from what they said was specific to HTLV-I, a retrovirus Gallo had earlier claimed to have discovered, and which they deemed suitable for this purpose. The DNA detected in this way was replicated and certain stretches of it cloned and declared to be the DNA of HTLV-III (later to be called HIV).
To summarise, the purpose of the exercise is to grow HIV, but it actually produces a mixture of different lengths of DNA, contrary to theory which says they should all be identical, and no virus at all. It is then claimed that the "correct" DNA has been prepared by finding certain strands in this heterogeneous mix by hybridising them with an HTLV-I DNA probe whose sequence is known and defined to be similar to HIV. However, non-hybridising strands of DNA should not be there at all, and the fact that they are, proves that a complete rag-bag of DNA has been prepared, without any indication of what it is made up of. �?BR>
Now the method of selection that Dr. Lanka is talking about here is different than what Bio lad is talking about. However what is important here is that Dr. Lanka has shown that random template switching can and does result in strings of cDNA that are very very similar to each other. Even though they are made from lots of cellular mRNA. This is not surprising as each mRNA that is used to make the cDNA is present in super abundance, millions and millions or them. Thus there are many opportunities to generate strings that are similar to each other. It just a game of chance. But since you are playing with so many millions and millions of copies of each mRNA you are garneted to get sequences that will be very similar to each other. Even greater than 99.3%.<o:p></o:p>
So according to Lanka random template switching does result in cDNA clones that are very similar to each other. But Bio lad says it does not. Who should I believe?<o:p></o:p>
Well both are clearly experts. Of coarse Dr. Lanka has a PhD in molecular biology and Biolad only has a masters. And Dr. Lanka has discovered and isolated several viruses in his carrier. As far as I know this is not rue of Bio Lad. If it was then he would have PhD like Lanka does.<o:p></o:p>
Also Bio lad has been caught hiding all kinds of really important stuff. Like the fact that template switching is used in making cDNA. <o:p></o:p>
He has also been caught in some major lies like saying template switching is can only be done with homology. Oh and also how he lied to me and said that there was only one cDNA, when in reality there is a whole soup of them.<o:p></o:p>
Yeah�?I think I’m going to put my trust in Dr. Lanka on this one, at least till I learn more molecular biology. I have been looking into many of Dr. Lanka’s ideas and so far they have always been right on.<o:p></o:p>
Ok, Dr. Lanka is the expert I trust on this one.<o:p></o:p>
Thus: template switching can and does result in highly similar cDNA clones that can then be fished out of the soup.<o:p></o:p>
Ok I am ready for bed now. There are a number of other things that Bio lad has claimed here but I am going to let other people on the board investigate that for now.<o:p></o:p>
However I have shown that Bio lad is distorting the facts as needed and hiding really important stuff from us.<o:p></o:p>
If Hep C was real he would not need to tell any fibs to prove its existence.<o:p></o:p> UNQUOTE. |
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