Techniques / Diagnosis Flashcards
Diagnosing Duchenne muscular dystrophy?
- multiplex PCR
- Western blot (detects presence and size of dystrophin protein)
Diagnosing fragile X syndrome
Southern blot
Screening… for neurofibromatosis type I
- sequencing; sanger method
Diagnosing sickle cell anemia
RFLP
restriction fragment length polymorphism
Diagnosing HIV
- ELISA
- Western blot to confirm the positive
What can FISH be used to diagnose?
- Prader-Willi syndrome
- Angelman syndrome
- Down syndrome
- CML
- ALL
What method is used for paternity test or fingerprinting?
VNTR (variable number tandem repeats)
What is ASO and what is its purpose?
ASO = allele specific oligonucleotides
–> Used to detect DNA mutations
What is karyotyping?
Assessing morphology of metaphase chromosomes
What is RT-PCR? (explain reverse transcriptase not real time here)
PCR applied to small sample of RNA. SS cDNA is synthesized from mRNA of interest with reverse transcriptase. Add PCR primer, then DNA polymerase
What is RT-PCR? (explain real-time here)
–> Quantify gene expression.
Used to measure amount of a DNA or RNA sequence in a sample relative to another control sample of DNA or RNA.
What is FISH? Explain it.
Fluorescence in situ hybridization.
Probes labelled with fluorescence-dyes contained within chromosomes immobilized on microscope slides to visualize chromosomal aberrations.
(Chromosomes either in interphase or metaphase; fixed i.e. in situ)
Steps of chromosome preparation
Indirect chromosome:
- take sample (G0) cells i.e. peripheral blood
- stimulate mitosis via adding PHA
- incubate 2-3 days
- stop mitosis @ metaphase by adding colcemid
- transfer to centrifuge, place cells in hypotonic solution, add fixative
- drop sample onto slide
- view under microscope, take picture
- use picture to cut out chromosomes and arrange in karyotype
