American Society of Cytopathology Core Curriculum in Molecular Biology

Transcription

1 American Society of Cytopathology Core Curriculum in Molecular Biology

2 American Society of Cytopathology Core Curriculum in Molecular Biology Chapter 3 Molecular Techniques Separation and Detection, Part 2 Marilee Means, PhD, SCT(ASCP) University of Kansas Medical Center Kansas City, Kansas

3 Objectives 1.Discuss the theory of in situ hybridization (ISH) 2.Define how FISH differs from ISH 3.Explain the methodology of detecting chromosomal mutations by FISH. 4.Describe the methodology used in Dot blot technique 5.Describe the methodology used in microarrays and its advantages

4 ISH - FISH In situ hybridization refers to the fact that this technique takes place in the cell or in situ. It is used to detect protein, RNA, and DNA. ISH is a more general term and may use various types of probes FISH (fluorescent in situ hybridization) uses a fluorescently labeled probe. Roche Diagnostics CD, Molecular Technology, Other Techniques, In situ hybridization

5 FISH This illustrates how the DNA in the chromosome of the cell can become separated when denatured, and bound to the probe which is labeled with a fluorescent dye. An advantage of FISH is that one can identify the exact chromosome which is affected. National human genome research institute

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7 FISH May be done on fixed cells and does not necessarily require the culture of live cells, as does traditional karyotyping. Each unique region on a particular chromosome should have exactly two signals, reflecting the 2 copies of the chromosome. Deletions or duplications (1 or more than 2 signals) reflect mutations in the chromosome Dual or tricolor probes may be used to increase the sensitivity of the test

8 FISH A metaphase cell positive for the bcr/abl rearrangement (associated with chronic myelogenous leukemia) using FISH. The chromosomes can be seen in blue. The chromosome that is labeled with green and red spots (upper left) is the one where the wrong rearrangement is present. The t(9;22) translocation of part of the ABL gene from 9 to chromosome 22 where it fuses with the BCR gene.

9 Whole Chromosome FISH Compared to Karyotype banding Whole chromosome paints can be used to detect small rearrangements not apparent by regular chromosome banding.

10 Dot Blot Used to detect single or multiple nucleotide changes without using restriction site alterations Does not require restriction digestion or electrophoresis Uses labeled, allele-specific, base oligonucleotide probes to detect mutation Will not tolerate a single base mismatch

11 Dot or Slot blot An example of the results of a dot blot analysis. To the left are two different sets of equipment for dot or slot blot.

12 Dot blot Target may be on the filter (standard) or the probe may be on the filter (reverse) Used to detect normal or mutant sequences (must know sequence) Can be used to detect sickle cell mutation in hemoglobin gene Normal codon is GAG and codes for Glu Abnormal codon is GTG and codes for Val

13 Sickle Cell mutation dot blot The dominant gene GAG, if present on both alleles, will mean the patient is normal and is not a carrier If GAG is present on one allele and GTG on the other, the patient is a carrier but does not have the disease If GTG is present on both alleles, the patient will have the disease

14 Sickle Cell Dot Blot

15 ASO = allele-specific oligonucleotide Allele-specific oligonucleotide (ASO) dot-blot hybridization can identify individuals with the sickle cell mutation The schematic dot-blot at top shows the result of probing with an ASO specific for the normal β-globin allele (βa-aso; shown immediately below). The results are positive (filled circle) for normal individuals and for heterozygotes but negative for sickle cell homozygotes (dashed, unfilled circle). The dot-blot at the bottom shows the result of probing with an ASO specific for the sickle cell β-globin allele (βs-aso; shown immediately above), and in this case the results are positive for the sickle cell homozygotes and heterozygotes but negative for normal individuals. The βa-aso and βs-aso were designed to be 19 nucleotides long in this case chosen from codons 3 to 9 of respectively the sense βa and βs globin gene sequences surrounding the sickle cell mutation site. The latter is a single nucleotide substitution (A T) at codon 6 in the β-globin gene, resulting in a GAG (Glu) GTG (Val) substitution (see middle sequences).

16 Microarray In 1987, treated glass was used instead of nitrocellulose or nylon membranes to produce a microarray Arrayers can deposit tens of thousands of spots on glass (such as a microscopic slide) Thus, the whole human genome can be deposited in triplicate on one slide called a chip

17 Microarray Targets are usually DNA, but may be other nucleic acids or protein Probes are usually cdna generated from sample RNA, but other nucleic acids or protein may also be used. Roche Diagnostics CD, Molecular Technology, Other Techniques, Microarrays

18 Microarray /dnaarray.html microarray.html Microarray-.html

19 Microarray Virtual Lab microarray/

20 Bead Microarray A newer microarray method uses 3 micron polystyrene or latex beads instead of a microarray on a chip to carry labeled probes. May be carried in an etched depressed on a slide on in liquid. Specificity is increased for closely related targets. Flow cytometry may be used on liquid specimens with microarray beads.