4 cellular Flashcards
What is the purpose of using the DNA polymerase chain reaction?
To amplify the number of copies of a specific DNA fragment—also useful as a diagnostic tool (as in neonatal HIV and herpes encephalitis)
A man presents with herpes infection. You want to know whether he has HSV-1 or -2. What molecular test can you use to differentiate the two?
DNA polymerase chain reaction
How is the size of a DNA fragment or polymerase chain reaction product determined from an agarose gel?
A ladder with DNA fragments of known lengths is run alongside the DNA fragment(s) of interest
How is DNA separated into two strands during polymerase chain reaction?
By heating (denaturation)
In polymerase chain reaction, premade ____ are used to anneal to specific DNA sequences that will then be amplified.
Primers—they bind to specific DNA sequences during cooling
What enzyme is used in polymerase chain reaction to amplify DNA?
Heat-stable DNA polymerase
What are the three steps of DNA polymerase chain reaction in order?
Denaturation, annealing, and elongation—these steps are repeated multiple times to amplify a DNA sequence
Describe the technique of a Western blot.
Gel electrophoresis separates sample protein that is then transferred to a filter, and labeled antibody is used to bind protein of interest
What blot technique detects specific sequences of DNA?
Southern blot (SNoW DRoP; Southern = DNA, Northern = RNA, Western = Protein)
What is the difference between a Southern and Northern blot?
Southern blot involves radiolabeled DNA probe binding to DNA, whereas Northern blot involves radiolabeled DNA probe binding to RNA
Describe the steps of a Southern blot after the DNA of interest is cleaved, electrophoresed on a gel, and transferred to a filter.
Filter is soaked in a denaturant, exposed to DNA probes that recognize and bind the DNA of interest & this DNA complex is then visualized.
____ blotting uses labeled oligonucleotide probes to identify DNA-binding proteins (such as transcription factors).
Southwestern
Which blotting procedures are useful for identifying transcription factors and studying specific gene expression levels in a sample?
Southwestern blotting; Northern blotting
A patient likely has HIV. Labeled antibody binds to relevant HIV proteins. After what screening test was this confirmatory test done?
An ELISA is done to screen and, if positive, followed with Western blot for confirmation
Which assay would allow you to detect single nucleotide polymorphisms or copy number variations to assess predisposition to a disease?
Microarrays—are also applicable for genotyping, clinical genetic testing, forensic analysis, cancer mutations, genetic linkage analysis
