Chapter 4 Flashcards
What is cytogenetics?
Provides an overall description of the chromosome #, structure, and extent and nature of chromosome abnormalities (above or less specific than at the genetic or molecular level)
Chromosomes are conventionally examined at WHAT phase of mitosis?
Metaphase spread
G bands are used to evaluate chromosomes for abnormalities. Give 1 example of where G band evaluation can ID a genetic abnormality. Are these evaluations harder in round cell tumors or solid tumors and why?
Detectable dark staining bands after separation of a metaphase prep. Examined by bright field microscopy and photos.
Can be used to ID translocations such as the Phila chromosome where there’s a translocation bt chromosomes. Also useful to ID extra chromosomes (trisomy), missing chrom, etc.
More difficult in solid tumors dt lower MI
How many chromosome pairs in dogs?
38 plus either XY or XX
What does it mean that DNA hybridizes?
How is this used to evaluate DNA in Southern blotting?
Hybridization: the matching of complementary DNA base to form a pair. DNA will only line up or bond w/ exact opposite or complementary match. Renaturation process after denaturing to form single strand of RNA.
In Southern blotting the DNA to be analyzed is cut into defined lengths using a restriction enzyme and fragments are separated by electrophoresis. Common application to determine size of a fragment of DNA that carries a particular gene (aka probe)
How does cDNA differ from regular DNA?
cDNA is the complementary DNA which is a copy of only the exons of the gene bc it is made using reverse transcriptase enzymes from mRNA. A copy of the useful genetic info.
What are restriction enzymes, how and where do they work, how is this useful in molecular genetics?
Restriction enzymes: endonucleases that cut DNA ONLY at sites of specific nucleotide sequences (t/f always cutting the DNA exactly at the same place w/in the sequence). For a given gene, the pattern for example on a Southern Blot of the DNA will always be the same if cut by a particular restriction enzyme. If the gene is mutated, the fragments after cutting by the enzyme will vary identifying a mutation is present
What product do these tests ID? So blot No blot Western blot Eastern blot
Southern - DNA
Northern - RNA
Western - Protein
Eastern - doesn’t exist
Does PCR increase the specificity or sensitivity of DNA detection?
How does PCR work?
How is RT-PCR different and what does RT-PCR stand for?
PCR increases sensitivity by amplifying DNA product to make it more likely to be detected.
Steps:
- Heat to denature (separate strands)
- Incubate at 53 allowing hybridization w/ new primers
- Incubate at 72 to allow TAQ to build new DNA strands…After 20 cycles = a million fold amplification
Uses TAQ polymerase to amplify DNA which is resistant to heat denaturation, primers specific for the DNA of interest, and many extra nucleotides.
RT = reverse transcriptase, used to make cDNA from mRNA to look only at coding parts of the gene and look for mutations
What is quantitative REAL-TIME PCR
used to quantitate low amounts of mRNA to quantify actual gene expression. Does this by using a fluorogenic probe for the detection of reaction products after PCR (ex. SYBR green I). Different dyes can be used simultaneously to analyze different probes. SYBR green only fluoresces when bound!!!! Very little fl. in unbound form.
NOT sequence specific - binds all DS DNA.
What are SNPs and how can they clinically cause an abnormality?
Single nucleotide polymorphisms.
Differences of DNA sequence at a single nucleotide. Occur 1 in 1000 base pairs. Occur in introns and exons but more nb in exons bc this can affect protein structure/function.
Can lead to missense mutations.
Detect w/ HPLC.
What is FISH (fluorescence in situ hybridization) and how does it work? How is M-Fluor different?
FISH - uses labeled DNA probes specific for a gene, chromosome segment or whole chromosome. They are hybridized to metaphase chromosomes labeling the precise location of the DNA on the chromosome. They are detected using AVIDIN which binds strongly to BIOTIN or fluorescing dyes. Good for detecting monosomy (only 1 copy), trisomy (3 copies), deletions if you have a probe for the deleted area, rearrangements (bcr-abl).
M-fluor uses multiple colors…
Explain SKY (spectral karyotyping)
Spectral karyotyping is based on the differential display of colored fluorescent chromosome specific paints which provide a complete analysis of the chromosomes in a cell.
A unique color is assigned to each chromosome pair. Easy to ID translocations.
Similar to M-fluor.
How do microarrays work?
Pertains to testing 1 sample for many different gene expression characteristics.
DNA mounted on a chip in 2 ways:
(1) DNA probe targets are immobilized to a solid surface and exposed to a set of fluorescently labeled sample DNAs - or -
(2) different oligonucleotide probes are made on the chip and exposed to a variety of labeled DNA samples.
Allows large scale gene discovery, expression, mapping in tumor types, and sequencing studies. Can be done w/ cDNA as well. Using different fluorescently labeled dyes, the expression of genes can be quantified. Fluorescent intensity correlates w/ gene expression. Gene expression can be clustered (4.17)
What is a tissue microarray?
Can take many paraffin embedded samples and analyze a number of different tumors or many patients w/ the same tumor for evaluation of expression of a particular gene. Up to several thousand tissues measuring 0.5-2mm in diameter will be mounted in one tissue array. One problem is that normal cells are often present in tumor samples, confounding results.
