Molecular Diagnos/cs I- DNA Flashcards
Open reading frame vs long stretches of genetic material
Only the coding region: w/ exons (introns spliced out). “Coding sequence of gene”
Huntington’s Disease
1st to be mapped
Autosomal dominant
Giant repeats on chromosome 4
SNPs
“typos” in replicated DNA strand
Can have no effect or can cause disease
Restriction endonucleases
Isolated from bacteria- cut DNA at specific sites (bacteria can rearrange genome). Ideally we want overhangs
Ligases
“gluing” DNA together
Much easier w/ compatible cohesive ends. Creates phosphodiester bond
Where? Inside nucleus on lagging strand
Restriction sites
5-6 base pairs that are very specific. If DNA endonuclease doesn’t cut what you expect, then maybe it’s not there
Parts of plasmid
(Usually) 2 restriction sites
Antibiotic resistance gene (AMP): stuff that doesn’t have our DNA won’t grow cause it needs AMP gene to survive in media
Origin of replication (where DNA polymerase binds)
cDNA
DNA copy of mRNA
- no introns
- much smaller than original gene
- requires reverse transcriptase
Steps of PCR
Heat, cool to anneal primers, replicate DNA
PCR applications
HIV: get RNA–>cDNA, amplify it and run on gel. p21 is usually what you are looking for
CSI: compare blood samples on gel. Utilizes naturally occuring short tandem repeats (STR), non-coding regions of repeat sequences (useful for paternity tests etc)
Looking for disease: if restriction enzymes don’t cut what you were expecting, there were probably mutations from a disease that messed up cutting spots
Microarrays
Can look at pathways and expression
Allows for thousands of genes to be identified at the same time
mRNA–>cDNA–>put onto microarray (both the normal cell and ‘cancerous’ cell will be hybridized and put onto microarray. The small DNA fragments will match up and bind to complimentary strands that are on the glass slide
Green=only normal cell
Red=only cancer cell
Yellow=both
Black=nothing
qPCR
Do PCR in presence of fluorescent dye. Only activated when samples binds to complimentary base pairs. Camera is above test tubes and you get signal that is corresponding to how much DNA was there. How much dye=how much DNA we started with.
Sample that needs less cell cycles to get to same fluorescent point tells us that it is over-expressing a certain gene
Lower Ct number (cycle number)=tells us it took less round of PCR to get same amount of signal so the original product of mRNA was in greater concentration
To measure the activity of every gene in a cell or tissue, use a DNA microarray chip
True
Restriction endonucleases are for DNA
True
Sickle cell= pt mutation causes loss of restriction site
True
