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On another newsgroup, http://groups.msn.com/aidsmythexposed/general.msnw, there was a debate about whether "Hepatitis C" virus had been isolated. I will quote a few passages below, and if anyone else wants to get involved, feel free, provided that you arguments are on point and supported by evidence. The posts below are from the "middle" of the debate, which involved others (not just the two quoted below, "Softrat" and "Biolad"):
Softrat:
QUOTE: Here they are:
http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=11314272&cmd=showdetailview&indexed=google
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=334354
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1370611
http://linkinghub.elsevier.com/retrieve/pii/S0014579302028120
http://www.pubmedcentral.nih.gov/articlerender.fcgi?&pubmedid=12584327
Look! What Dr. Lanka was talking about!
"When RT switches templates during cDNA synthesis, it creates a progeny cDNA sequence that harbors sequence identity to both templates"
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WG1-4J5T5V7-1&_user=10&_coverDate=07%2F31%2F2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=5792be01e26c83d47188af2c0f4b8d17
This stuff is all over the net! Just use your search engines.
Bio Lad said:
"First, a cDNA is a DNA copy generated from an RNA template. You must therefore have an RNA template to begin with. To claim they generated cDNA without having isolated RNA is just silly. It is like saying you made a photocopy of a page without using the original paper"
Yes many little mRNAs you have isolated and then template switched.
"They then reverse transcribed the RNA and THAT is how they made their cDNA. The cDNAs they obtained are nothing more than DNA copies of the RNAs they isolated. "
But cDNA is made from template switching. Like I said.
"Nope, once again it is your misunderstandings and assumptions that lead you to believe that. Especially since the paper describes the RNA isolation (M&M, paragraphs 1 and 2)."
Once again cDNA is made from template switching. UNQUOTE.
And:
QUOTE: This is fromthe study Biolad cited:
"A lambda gt11 cDNA library was constructed from RNA purified from hepatitis B viral surface antigen-negative human plasma with high alanine aminotransferase activity. A cDNA clone, designated as C8-2, was isolated by immunoscreening with mixed sera from non-A, non-B hepatitis (NANBH) carrier and convalescent chimpanzees"
Now look at the quotes in the above post. Pay note to this one:
"Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step."
It looks like template switching is how cDNA is made. UNQUOTE.
Biolad:
QUOTE: SoftRat,
Your reasoning on the issue of template switching is not as relevant as you would think. I am in the process of typing up a full rebuttal using, amongst others, the citations you provided. Since, however, you seem to have a history so far in this debate of making assumptions based on misunderstandings, please take this time to look back through your sources and try to see if you can figure out why your argument it not valid. I should have my post sent tonight as I am busy at work today. Keep in mind that my posts all go through Rod first so it may take a while to actually get posted so they generally have a bit of a lag between the time I send it and the time it gets posted. It is understandable though as I'm sure he has other things to do as well.
-BioLad UNQUOTE.
Softrat:
QUOTE: What we have learned Part 2
<o:p></o:p>
By: The Soft Rat
<o:p></o:p>
Ok very interesting stuff.
<o:p></o:p>
Bio tried to argue that I was wrong about cDNA being made via the template switching method. But it turns out I was in fact completely right about this. He also claimed that Dr. Stephan Lanka, some with a PhD in molecular biology and who has isolated his own phage virus old school style, was wrong about viral cDNA being made artificially through reverse transcriptase template switching.
<o:p></o:p>
But as these quotes show Bio Lad appears to have been misleading us. Remember Bio Lad has a masters in molecular biology so he is well aware that cDNA is made via reverse transcriptase template switching. Which means that him trying to mislead us this way is no honest error on his part.
---------
The Internet source for these quotes are listed in above post and it is easy to find this stuff on the net with a search engine:
<o:p></o:p>
“When RT switches templates during cDNA synthesis, it creates a progeny cDNA sequence that harbors sequence identity to both templates"<o:p></o:p>
<o:p></o:p>
Real important one here as this is not only what Dr. Lanka was talking directly about but it shows that cDNA is made from many small pieces of mRNA.<o:p></o:p>
<o:p></o:p>
“cDNA strands produced from template switching were selectively amplified�?lt;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>
More proof that cDNA is made via the template switching method.<o:p></o:p>
<o:p></o:p>
This next one is important as it shows that template switching creates fake genomes:<o:p></o:p>
<o:p></o:p>
“Evidence that BmTXKβ–BmKCT cDNA from Chinese scorpion Buthus martensii Karsch is an artifact generated in the reverse transcription process. "<o:p></o:p>
<o:p></o:p>
<o:p></o:p>
“Because it is known that reverse transcription (RT) can produce artefacts by strand switching during the RT step�?�?BR>
<o:p></o:p>
This last quote is from the scorpion paper at:
<o:p></o:p>
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1370611
<o:p></o:p>
“Here, we have investigated the presence of such template-switching artifacts in
cDNA databases�?lt;o:p></o:p>
<o:p></o:p>
<o:p></o:p>
So cDNA is made via template switching that does create cDNA genomes that do not correspond to real world genomes. It creates fake genomes.
<o:p></o:p>
Bio Lad didn’t tell us any of this. He clearly did everything he could to steer us away from template switching and deny that the Hep C cDNA was made via template switching. This is no over site on the part of Bio Lab. He has a Masters in molecular Biology. He knows exactly what he is doing here.
<o:p></o:p>
The critical and fundamental part of my argument is that the cDNA of Hep C is made via template switching from smaller unrelated mRNA molecules. And therefore the Hep C mRNA is not found in nature but is a Lab artifact.
<o:p></o:p>
Take the time to read through the study Bio Lad sited. There is not one shred of evidence that they isolated a single molecule of mRNA and somehow made the cDNA from that. Instead if you read the materials and methods section you will see the just extract out mRNA from the plasma. There is no proof the Hep C mRNA is in there as a single entity. From there they make cDNA via reverse transcription. And as the evidence above shows cDNA is made via template switching with reverse transcription enzymes. Just like I was saying.
<o:p></o:p>
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=330752&blobtype=pdf
<o:p></o:p>
<o:p></o:p>
Now Bio Lad said:
<o:p></o:p>
“They then reverse transcribed the RNA and THAT is how they made their cDNA. The cDNAs they obtained are nothing more than DNA copies of the RNAs they isolated�?lt;o:p></o:p>
<o:p></o:p>
Notice how tricky he is. He tells lies of omission. He doesn’t deny the template switching directly, he just fails to mention it. However given the fact that he is rebutting my template switching argument he is clearly imply that template switching is not happening. But cDNA is made by template switching.<o:p></o:p>
<o:p></o:p>
“Nope, once again it is your misunderstandings and assumptions that lead you to believe that. Especially since the paper describes the RNA isolation (M&M, paragraphs 1 and 2)."<o:p></o:p>
<o:p></o:p>
Once again he fails to mention that they extract or isolate mRNA, but there is no proof that the mRNA they extract contains any mRNA existing in the form of the Hep C virus. And given the fact that they then make cDNA which is made via template switching it is clear that the sample must not have. The extracted sample must have been composed of many small pieces of mRNA that were then template switched together into the cDNA.<o:p></o:p>
<o:p></o:p>
Now if Hep C virus really does exist why is Bio Lad, someone who has a masters in molecular biology, misrepresenting the way cDNA is made? Why is doing everything in his power to steer us as far away as possible from understanding the fact that cDNA is made via template switching with reverse transcriptase enzymes?<o:p></o:p>
<o:p></o:p>
Remember my argument that Hep C mRNA does not exist, and thus Hep C virus does not exist is based on the fact that cDNA is made via template switching. The very thing Bio Lad tried to mislead us into believe was not true. I think the facts speak for themselves.<o:p></o:p>
<o:p></o:p>
Also do not forget that Bio Lad failed to inform us that when the human, or chimp, DNA genome database is made they automatically remove any and all evidence of viral genomes in there. So this is why when you do a BLAST nothing shows up. Once gain why did Bio Lad fail to mention this to us?<o:p></o:p>
<o:p></o:p>
I think Bio Lad is someone that came here to stop all this talk about viruses not existing. I don’t know if he came here on his own, or if some company, or the NIH sent him or what. But it is clear that this is what he is trying to do. It is also clear that he will distort the evidence as needed to do this.<o:p></o:p>
<o:p></o:p>
This is not consistent with Hep C or viruses in general exist. If they did exist Bio Lad could reveal all relevant data on the subject matter and let reality speak for itself.
------
Update:
Bio lad said this:
"Your reasoning on the issue of template switching is not as relevant as you would think."
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?
Bio lad you are hiding relevant information here. As a molecular biologist you should not have to do that if Hep C virus really does exist. UNQOTE.
And:
QUOTE: Bio Lad:
I told Rod Knoll I would stick with you till Monday. When I get home tonight it will be Tuesday Morning. So this will be my last post on this Hep C series.
You are now about to construct an argument to show that my reasoning on template switching is not as relevant as I think it is.
However you felt no need to construct any such argument until I revealed that you had been hiding the fact that cDNA is in fact made via template switching via reverse transcription enzymes. This clearly shows that you where covering up that the Hep C genome is created from smaller mRNA sequences strung together via template switching. Now that I have showed this is the case you will cinstruct an argument that shows this is not really as relevent as I think it is.
Bio Lad in order for you to be regarded as a valid expert on molecular biology, and the existance or non-existance of Hep, we have to be able to trust what you say. We can't regard you as a reliable witness if we have to worry about if you are telling us everything we need to know in order to come to a relaity based conclusion.
As an expert in molecular biology, and a researcher, you have hidden two very imporatnt things that you would clearly have known about. One is that I was dead on the mark about cDNA being made via template switching. The other that the reason Hep C sequences can not be found in the data base when you BLAST is because they would have been removed, or never added, from the data base when they were found.
Because you have hidden these things is does not matter what you say in your next rebuttle. You simply can not be trusted. If I looked through your rebuttle and worked long and hard on it I might be able to uncover how you are fibing or hiding something important and then I would post that here. The only problem is you would then construct a new argument showing how this new information I have uncovered, and that you were hiding, is not realy as relvant as I think it is. I do not care to find out how long you would be willing to carry this on.
I have had to put aside other things I would rather be working on in order to have this debate. The fact that you have been hiding the fact that cDNA is made via template switching means you have been wasting my time. The fact that you do not reveal relevant facts, and pretend they do not exist, and only then deal with them after I show you have been hiding them means your testimony about Hep C carries a value of zero epistemologically. If I was on the jurry in court I would have to disregard your testimony as there is no way for me to know when you are telling the truth or not. No way for me to know if you are telling me everything I need to know to come to a valid conclusion.
Also lets look at this:
"First, a cDNA is a DNA copy generated from an RNA template. You must therefore have an RNA template to begin with. To claim they generated cDNA without having isolated RNA is just silly. It is like saying you made a photocopy of a page without using the original paper. Second, The Chiron group did in fact isolate the RNA from serum. They state it quite clearly in the first page under materials and methods (second paragraph). After obtaining the RNA they state:
"The RNA was further purified by DNase treatment (1. 15KU/mlDNase, 50 mM Tris-HCl pH7.4, 1 mM EDTA, 10 mM MgCl2)
for 30 min at 37°C, and anion exchange chromatography (Qiagen pack-100, Diagen)."
Since you seem sketchy on your molecular biology, and for the benefit of anyone not familiar with it, DNAse is an enzyme that degrades DNA into single base units. This means that any DNA that may have been present in the serum is now completely degraded and no longer usable as a template. They then reverse transcribed the RNA and THAT is how they made their cDNA. The cDNAs they obtained are nothing more than DNA copies of the RNAs they isolated. (btw, in case you are about to make another bad assumption, the DNAse is heat inactivated prior to reverse transcription.)"
Notice you never directly say that the cDNA is not made via template switching. You just fail to mention it. You fail to mention it in this post or any others even though you know I have been saying the cDNA is made via template switching. You fail to mention this fact in any post even though you know the template switching is the fundamental essense of my argument that the Hep C genome is made from smaller mRNAs strung together in template switching, and thus the Hep C mRNA genome does not exist in nature.
You only now want to deal with this fact after I have shown that cDNA is made via template switching. Befoe this you were more tha happey to keep this under wraps. You are not a trustworthy source of informationon this isuue Bio Lad. There is no way anyone can know when they read yournext post if you are being honest with them or not. Thus your testimony must be thrown out of court.
I do not know what else you may have hidden from us that I or others have not yet uncovered. But let me tell you this Bio Lad. I have dusted off my old college BioChem text book. Since this was written some time ago I will be going to the UW and also buying a new text on molecular biology so I can have up to dat information. So given a few years I will catch up to you.
Also like I told you I am studying virology from begining to end. Oneof the reasons I am doing this is cause the early research on virology is on my level. As I move through the papers I learn the methods of molecular biology as the scientist of those days developed them. So I learn as they learned. In the order that the methods were developed.
I also get to see how the belief in replicating particles started. I get to read the papers from those days that reported evidence against virology. I and guess what Bio Lad? In those days there were scientist that disagreed with the beleif in viruses. There where scientist that showed that the disease causing agents did not self replictae. That the sap of plant viruses did loose potency as it was transfered from pant to plant. That high doses of that sap produced strong effects, and low dosses produced weak effects, just like a finite toxin would. I am working on a paper write now that goes into all of this and documents all the research papers on this matter. So given a couple years I will get back to Hep C. UNQUOTE.
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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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One of my posts in the the thread from the other newsgroup contained the following quotation:
"It is therefore possible that the GOR gene product is a nuclear protein that contributes to the development or regulation of hepatitis. Furthermore, we (unpublished observation) and Dennin et al. [18] found a conserved homologous gene in other animals and plants, suggesting that the GOR gene might be universally important and has basic physiological roles. Our ongoing studies include analysis of the physiological roles of the GOR gene product in an attempt to fully elucidate the relationship between the gene and HCV."
Source: Clin Exp Immunol. 2001 June; 124(3): 429�?34 is at:
On the internet: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1906082
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Another important passage from that study I cited in the post above is:
"The 5'-NCR of the HCV-RNA acts, through an IRES, as the starting area for translation. However, the respective sequence sections present in the DNA may serve as a potential source of transcriptional activity. This can be explained in terms of ongoing DNA rearrangements(19,20) and the exchangeability of IRES units(21) that result in chimeric transcripts. Our data would further indicate that the sequence stretches of up to 272 base pairs detected by PCR could be present in some kind of extra-chromosomal 'rearranged DNA' (belonging to mobile DNA elements like DNA transposons). This interpretation is supported by a report on expressed-sequence tag (EST) libraries.(22) It demonstrates that a human RNA library already contains a sequence stretch (286 nucleotides with 92.7% homology) of the HCV genome that is not contained as such in human DNA libraries." |
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