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Pcr product size determination for a fusion gene


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8 replies to this topic

#1 de ifz

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Posted 26 August 2012 - 11:18 AM

Hi there!

I am facing a serious problem as I want to amplify a "Fusion oncogene" causing Chronic myeloid leukemia CML. I have got primer sequences from a research paper and it worked. The PCR is positive.

PROBLEM is that I don't know the exact size of my PCR product. Just roughly know that it is about 1.2 kb.

I tried NCBI blast . many hit and trials but ....

my Forward primer belongs to one gene i. e BCR gene on chromosome 22

and REVERSE primer belongs to the other gene i. e ABL on ch 9.

(These two genes combine to cause leukemia...fusion oncogene)

NCBI doesn't recognize both primers together, but blasts them separately to their respective genes.

Help please.
worried alot :(

#2 bob1

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Posted 26 August 2012 - 12:33 PM

Probably the easiest way to determine the full lenght will be to sequence the product you have and/or clone it, then sequence.

However, just found this paper:
Vaccination of patients with chronic myelogenous leukemia with bcr-abl oncogene breakpoint fusion peptides generates specific immune responses

J. Pinilla-Ibarz, K. Cathcart,T. Korontsvit,S. Soignet,M. Bocchia,J. Caggiano,L. Lai,J. Jimenez,J. Kolitz, andD. A. Scheinberg

Which contains the following paragraph (second one in the paper, immdeiately following the abstract, bolding mine):
"Chronic myeloid leukemia (CML) is a pluripotent stem cell disorder characterized by the presence of the Philadelphia chromosome (Ph1). Ph1 is the result of a translocation of the c-abl oncogene from chromosome 9 to the breakpoint cluster region (bcr), within the bcr gene on chromosome 22, forming a chimeric bcr-abl gene.1-3 The fused genes encode an 8.5-kb chimeric mRNA that is translated to a 210-kd protein.4-6 This p210 bcr-abl protein shows tyrosine kinase activity, is present in the leukemia cells of patients with CML, and is necessary and sufficient for transformation.7 In 95% of patients, the breakpoint in the bcr gene occurs either between bcr exon 2 (b2) and 3 (b3) or between bcr exon 3 (b3) and 4 (b4). Hence, 2 alternative chimeric p210 bcr-abl proteins, comprising either a b3a2 or a b2a2 junction, can result from this fusion gene.8"

#3 de ifz

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Posted 28 August 2012 - 11:04 AM

thanx bob for the post...

the 8.5 kb size is actually encoding the whole cancer causing gene. suppose following is the fusion of BCR-ABL gene 8.5KB

123456789101234567
I am amplifying like

6789101234

for this I am unable to determine the size. I can roughly say that my product on gel appears to be 1.2 kb........??

The point mutations I am looking for belong to 1234 part. For which I got another set of primers. I got it sequenced and its size is 780 bp.

any further help plz.....
.

#4 ascacioc

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Posted 28 August 2012 - 11:41 AM

Is it 780 bp with one primer? Or is it 780 with fwd and rev sequencing? The sequencing (as it is in the sequencing companies) is limited to 800-1000 bp. Moreover, don't sequence with the same primers you are doing the PCR, see here an explanation why:

http://www.protocol-...-ligation-help/

Better clone it in a plasmid and then sequence from the backbone of the plasmid towards the insert.

Andreea

#5 bob1

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Posted 28 August 2012 - 03:18 PM

suppose following is the fusion of BCR-ABL gene 8.5KB

123456789101234567
I am amplifying like

6789101234

If you know which exons you are using and the size of the exons (try the BLAT tool at UCSC for getting the exons), can't you just add them up?

#6 de ifz

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Posted 30 August 2012 - 11:45 AM

Is it 780 bp with one primer? Or is it 780 with fwd and rev sequencing? The sequencing (as it is in the sequencing companies) is limited to 800-1000 bp. Moreover, don't sequence with the same primers you are doing the PCR, see here an explanation why:

http://www.protocol-...-ligation-help/

Better clone it in a plasmid and then sequence from the backbone of the plasmid towards the insert.

Andreea

Yes it is 780bp with forward and reverse seq. Lac of chemicals and limited budget of my project doesnt allow me the plasmid option :(

#7 de ifz

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Posted 30 August 2012 - 11:49 AM

If you know which exons you are using and the size of the exons (try the BLAT tool at UCSC for getting the exons), can't you just add them up?


I know the exons...ok I try the adding up option...lets see if it works

#8 ascacioc

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Posted 30 August 2012 - 01:08 PM

I know the exons...ok I try the adding up option...lets see if it works


This is exactly what I wanted to suggest right now :)

#9 Trof

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Posted 31 August 2012 - 06:44 AM

You can't BLAST fusion gene unless his complete sequence is in database. There are sequences for different BCR/ABL fusions but they are only short and encompass the fused region.

There are three main forms of CML transcripts, caused by breakpoint in three sites major, minor and micro.
You are probably working with the major breakpoint, which is most common.
Most common transcript of this breakpoint is designated e1a2, which means ex1 of BCR and ex2 of ABL (these are connected, the breakpoint is in intron, so these are joined complete exons). But there are some alternate transcripts and alternate exons, so the nomenclature is bit confusing.
Use this paper to navigate between different BCR/ABL fusion genes.
Attached File  Standart RT-PCR fusion transcripts in acute leu.pdf   370.58KB   554 downloads

All papers apart, easy way to find what transcript you have (unless you sequence) is to find BCR transcript on Ensembl, in Exon view, and find in which exon you have your primer.
Do the same for ABL and reverse primer.
Now you know there must be only whole exons between your primers, and you can mostly have fusions only between 1st (major), 13th or 14th (minor), 19th (micro) exon of BCR and 2nd or 3rd of ABL and these combinations.
I think there may be different very rare breakpoints apart from those in BCR but not in ABL.


The paper you got primers from though, should specify what they amplify.

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