Construction of Reference Plasmids for The Quantification of Bcr-Abl Fusion Transcripts in Chronic Myeloid Leukemia By Quantitative Real-Time Pcr

Abstract

Chronic myeloid leukemia (CML), is a myeloproliferative disorder characterized by rapid increased

proliferation of the granulocytes. The hallmark of CML is the presence of the Philadephia chromosome,

which is a product of translocation between the chromosomes 9 and 22 and as a result, a fusion gene

is formed between the BCR gene (chr. 22) and ABL (chr.9). Several variants of the BCR-ABL fusion

transcript exist which is based on the different breakpoint locations between the BCR and ABL genes.

The most known variants are designated p190, p210 and p230 fusion transcript. Since the amount of

BCR-ABL fusion transcript is correlated with the tumor load, therefore is important to quantify the

expression of BCR-ABL in the patient samples. For p210 fusion transcript, a reference plasmid was

created and the quantification was facilitated by having a standard curve made by serial dilution of

the reference plasmid. Unfortunately, reference plasmid for p190 and p230 remain absent. Here in this

study, we designed and construct two reference plasmids for quantification of the p190 and p230 fusion

transcripts. Unfortunately, the construction of the reference plasmid for p190 was unsuccessful,

after ligation, none of the colonies analyzed contained the 1000-bp insert of the p190. In contrast, the

p230 reference plasmid, named pBL-230, was constructed in which contain a 620-bp fragment of the

p230 fusion transcript. We showed that serial dilutions of pBL-230 could be accurately quantified by

two different UPL probes and can be used for quantification of p230 fusion transcript in CML patients.

Keywords

• Chronic myeloid leukemia

Kronik Myeloid Lösemide BCR-ABL Füzyon Transkriptinin Eş Zamanlı PCR ile Kantitasyonu İçin Referans Plazmid Oluşturulması

Abstract

Kronik Myeloid Lösemi (KML), granülositlerin hızla artan proliferasyonuyla seyreden bir myeloprolifertif

hastalıktır. KML, 9 ve 22 kromozomları arasındaki bir translokasyonla oluşan Philadelphia kromozomunun

varlığı ile karakterizedir ve bu translokasyon sonucunda BCR (22. kr) ve ABL (9. kr)

arasında bir füzyon gen meydana gelir. BCR ve ABL genlerindeki kırık bölgelerine bağlı olarak farklı

BCR-ABL füzyon transkriptleri oluşmaktadır. En bilinenleri p190, p210 ve p230 füzyon transkriptleridir.

BCR-ABL füzyon transkript miktarı tümör yükü ile doğrudan ilişkili olduğu için hasta örneklerinde BCRABL

anlatımını sayısal miktar olarak belirlemek önemlidir. Daha önce p210 transkripti için bir referans

plazmid oluşturulmuştur ve bu plazmidin seri dilüsyonları ile elde edilen standart eğrisi ile sayısal

miktar tayini yapılmaktadır. Ancak maalesef benzer bir plazmid p190 ve p230 kırıkları için mevcut

değildir. Bu çalışmada hem p190 hem de p230 transkriptlerinin miktar tayinini yapabilecek bir

referans plazmid oluşturmayı amaçladık. Ancak p190 plazmidi bütün uğraşlarımıza rağmen başarıya

ulaşamadı, elde ettiğimiz kolonilerin hiç biri p190’ın 1000bç’lik fragmanını içermiyordu. Bunun aksine

p230 referans plazmidi (pBL-230) 620bç’lik p230 fragmanı ile klonlanabildi. pBL-230’un seri

dilüsyonları ile iki farklı UPL prob kullanarak KML hastalarında p230 transkript miktarını tayin

edebildik.

Keywords

• Kronik Myeloid Lösemi (KML)

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