ABSTRACT
ALLELE-SPECIFIC OLIGONUCLEOTIDES
●● Probes can be constructed to differ by only one nucleotide
●● One probe recognizes the normal allele, and the other will recognize the mutant allele
DNA MICROARRAYS
●● cDNA is used to identify and quantify the genes of interest
●● Patient’s DNA is labeled in green and the same quantity of control DNA is labeled in red
●● Mixture is then hybridized onto the microarray
●● If equimolar quantities of test and control DNA are present, there will be a yellow signal (equal combination of red and green)
●● If the test DNA is in excess, signal will be green (trisomy 21, N-MYC amplification)
●● If the control DNA is in excess, signal will be red (del p53, del 5p syndrome)
GENE EXPRESSION MICROARRAYS
●● Determine the degree of expression of genes of interest
●● DNA is digested with DNAse → residual RNA converted to cDNA using reverse transcriptase → cDNA is labeled in green → hybridized onto microarray → intensity of signal correlates with gene expression
SINGLE NUCLEOTIDE POLYMORPHISMS
●● Study the differences involving only one nucleotide in a specific sequence, with probes specifically identifying these variations
●● Assay of the whole genome through microarrays, rather than studying a single gene on a membrane (differential diagnosis [d/d] ASO)
●● Useful for assays of trisomy, monosomy, and loss of heterozygosity (LOH) in tumors
FLUORESCENT IN SITU HYBRIDIZATION
●● Identifies segments of chromosomes spread onto a slide using probes
●● Chromosomes can either be in metaphase or interphase
●● Quantitative fluorescence in situ hybridization (FISH) to determine the number of genes in a cell
translocation partners are anticipated
●● BCR-ABL translocation of Philadelphia chromosome in leukemia
BREAK-APART PROBES
●● Series of two probes, each labeled in a different color
●● In oncogenic translocation, one probe hybridizes to one segment immediately above the break point, and the other hybridizes immediately below that locus
WHOLE CHROMOSOME PAINTING
●● Multiple same color probes used to hybridize different portions of the entire length of a pair of chromosomes
●● Metaphase spread is visualized and translocations studied
SPECTRAL KARYOTYPING
●● Every chromosome is painted with a different color probe (22 colors for autosomes, 1 color for X chromosome, 1 color for Y chromosome)
COMPARATIVE GENOMIC HYBRIDIZATION
labeled in green ●● Both DNAs mixed in equimolar concen-
trations, hybridized to normal metaphase spread (chromosome-based CGH) or chips (chip-based CGH)
●● Surplus of red signal in a chromosomal region = duplication in that region
●● Surplus of green signal = deletion in that region
●● Chip based better (cheaper, faster, and more sensitive) than chromosome based
NEWER GENERATION CHIPS
●● Combine standard CGH (comparative genomic hybridization) and SNP (single nucleotide polymorphism) probes
●● Used and being developed for a large variety of diseases
●● Will revolutionize clinical genetics, oncology, pharmacology, and pathology in the future
NORTHERN BLOTTING
or active in a tissue, gene expression profiling, etc.
