Molecular Diagnos/cs I- DNA Flashcards Preview

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Flashcards in Molecular Diagnos/cs I- DNA Deck (15):
1

Open reading frame vs long stretches of genetic material

Only the coding region: w/ exons (introns spliced out). "Coding sequence of gene"

2

Huntington's Disease

1st to be mapped

Autosomal dominant

Giant repeats on chromosome 4

3

SNPs

"typos" in replicated DNA strand

Can have no effect or can cause disease

4

Restriction endonucleases

Isolated from bacteria- cut DNA at specific sites (bacteria can rearrange genome). Ideally we want overhangs

5

Ligases

"gluing" DNA together

Much easier w/ compatible cohesive ends. Creates phosphodiester bond

Where? Inside nucleus on lagging strand

6

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

7

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)

8

cDNA

DNA copy of mRNA

-no introns
-much smaller than original gene
-requires reverse transcriptase

9

Steps of PCR

Heat, cool to anneal primers, replicate DNA

10

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

11

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

12

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

13

To measure the activity of every gene in a cell or tissue, use a DNA microarray chip

True

14

Restriction endonucleases are for DNA

True

15

Sickle cell= pt mutation causes loss of restriction site

True