Genetics E1- Diagnostic Methods

What type of cytogenetic testing produces a visible karyotype & identifies pairs of chromosomes and banding patterns?

G banding

What kind of tissue can G (Giemsa) banding be used on?

Any tissue containing nucleated cells that under go division- lymphocytes (MC), skin, BM, chorionic villi, amniotic fluid, etc

How does G (Giemsa banding) work?

Chromosomes are fixed, mounted on a slide, & stained which reveals distinct bands and allows for analysis of chromosome structure

What type of cytogenetic testing identifies and maps chromosomal & gene abnormalities, and identifies where a particular gene falls within an individual’s chromosomes?

FISH

How does fluorescence in situ hybridization (FISH) work?

Short sequences of single stranded DNA (probes) match a portion of the gene of interest → determines degree of sequence identity & detects specific sequences

What are examples of medical applications for FISH cytogenetic testing?

Detect aneuploidy, loss of chromosome or region, detection of specific cancers (HER2, bladder, leukemia), & monitoring success of BM transplantation

What type of DNA testing is used to amplify DNA sequences to look for genetic variants known to cause certain diseases?

PCR

How does polymerase chain reaction (PCR) work?

Short DNA sequences (primers) select the portion of the genome to be amplified → temp is raised & lowered repeatedly to help a DNA replication enzyme copy the target sequence

What DNA testing technique uses a special chemical that marks each DNA nucleotide with a different colored fluorescent dye depending on the base it carries?

Sanger sequencing

What is the limitation of Sanger sequencing?

Can only read one short section of DNA at a time

What type of DNA testing is much faster & sequences millions of small DNA fragments simultaneously?

Next generation sequencing (NGS)

What utilizes techniques to examine the protein instead of the gene, determining if certain proteins are present or absent & looks for enzymatic defects?

Biochemical analysis

What can biochemical analysis be used to detect?

Inborn errors of metabolism (PKU, CF), measures protein activity, level of metabolites, and size & quantity of protein

What uses comparative genomic hybridization to report on gains or losses throughout the genome, can detect smaller gains/losses than traditional analysis, & detects specific types of mutations?

Microarray

What type of mutations does microarray primarily detect?

Large genomic rearrangements (deletions/duplications) in genes like BRCA1 & BRCA 2

What risks does microarray carry?

Possibility of not detecting triploidy (depending on array type) & finding variants of uncertain significance

What are benefits of direct-to-consumer genetic testing, which are sold online & in shops?

Can provide info about risk/carrier states, gives a better sense of personal health agency/motivation to screen, & can be less expensive and faster

What are limitations to direct-to-consumer testing?

Often doesn’t test for pathogenic variants for some conditions, may report on conditions w/o confirmed medical utility (ex-which vitamins to take), false + & -, pt may not understand implications of results, reveal distressing ancestral relationship, & law enforcement may access to solve crimes

How is direct-to-consumer testing limited when evaluating BRCA genes?

Only examines 3 genetic variants, but there are more than 1000 mutations known to affect risk of breast & ovarian cancer