Lecture Sixteen

Chromosomal translocation-
Philadelphia chromosome
 Chromosomal translocations

• In a translocation, a segment from

one chromosome is transferred to
a non-homologous chromosome
or to a new site on the same
chromosome.

• Translocations generate novel

chromosomes.

• Translocations place genes in

new linkage relationships.
 Chimeric BCR-ABL gene (Philadelphia chromosome)

• Philadelphia chromosome is a product of a reciprocal translocation involving

small segments at the ends of the q arms of chromosomes 9 and 22.

• Philadelphia chromosome was initially identified in patients with chronic

myelogenous leukemia (CML).

• Subsequent molecular analyses revealed that the translocation fused the

coding sequence of the BCR (breakpoint cluster region) gene on chromosome
22 with the coding sequence of the ABL gene on chromosome 9.

• Replacement of the first ABL exon with sequences from BCR results in deregulation
of the tyrosine kinase activity of the normal ABL product.
Detection of chimeric BCR-ABL gene
 Multi-Color FISH to check for translocations

Processed by a computer
Image obtained in
to generate a
the microscope
false-color image

• This is an example of a spectral karyotype prepared from acute myeloid

leukemia cells with multiple translocations, which are indicated by arrows.

• This figure shows portions of the

karyotype in which translocation
chromosomes were placed next
to their normal homologues.
 Detection of chimeric BCR-ABL gene

• Traditionally and most commonly, western blot analysis (also called

immunoblotting) is the most powerful protein quantification method.

• Western blotting can provide sensitive and selective detection and quantitative
information

• But its general use is sometimes hampered by a high level of background signals
when detecting proteins at very low concentrations, time-consuming and
complicated experimental process, and necessity of a trained expert.
 Detection of chimeric BCR-ABL protein

“Here, we introduce a protein detection and quantification

method with a rapid and simple experimental protocol that
retains the selectivity and sensitivity of western blotting”
Detection of chimeric BCR-ABL protein
 Detection of chimeric BCR-ABL protein

• This approach takes only a couple of hours, except for formation of the
self-assembled monolayer and antibody immobilization on the AuNPs and
gold chips.

• These steps do not require a high degree of skill.

• Furthermore, our approach affords substantially low background signals

and false-positive signals, resulting in enhanced selectivity.
Detection of chimeric BCR-ABL gene
 Massive parallel sequencing-
Illumina sequencing

Preparation of genomic DNA sample

Attachment of DNA to surface
Amplification of attached DNA fragments
Amplification of attached DNA fragments
Amplification of attached DNA fragments
Amplification of attached DNA fragments
Sequencing of amplified fragments
Sequencing of amplified fragments
Sequencing of amplified fragments
Sequencing of amplified fragments
Sequencing of amplified fragments
Hybridization assays for detection of Ph chromosome
Time-resolved immunofluorometric hybridization assays for
detection of Ph chromosome

• Two hybridization assays have been developed to detect BCR-ABL mRNA

transcripts arising from the Philadelphia translocation.

• Both assays use time-resolved immunofluorometric detection of

polymerase chain reaction-amplified BCR-ABL mRNA sequences
hybridized to specific probes.
Time-resolved immunofluorometric hybridization assays for
detection of Ph chromosome

• In configuration I, biotinylated amplified target is immobilized onto

streptavidin-coated wells and hybridized to a probe labeled with
digoxigenin.

• Hybrids are detected via an alkaline phosphatase-labeled antibody.

 Time-resolved immunofluorometric hybridization assays for
detection of Ph chromosome

• In configuration II, biotinylated probe is immobilized onto streptavidin-

coated wells.

• PCR, performed in the presence of Digoxigenin-labeled

deoxyribonucleotide, generates labeled product, which is hybridized to
immobilized probe and quantified as above.

• BCR-ABL transcripts from one leukemic cell amidst mRNA from 500,000
normal granulocytes are detectable.