Molecular Diagnostic methods Part B Flashcards
an oligonucleotide designed to bind near a portion of the nucleic acid that is desired to be amplified (the target); two of them are needed and must be complementary to opposite strands flanking the target sequence
primer
artificial replication; important when the nucleic acid of interest in a clinical sample is very small (viral and bacterial infections)
amplification
a short single-stranded segment of nucleic acid
oligonucleotide
In vitro molecular diagnostic techniques function in detecting particular sequences in _____ ______
nucleic acids
If you use anticoagulated whole blood in testing it cannot contain what?
heparin
List the steps in a typical DNA extraction process (4)
1 Lyse cells: incubate with detergents and protease K
2 Isolate the DNA
3 Purify the DNA (multiple washing steps)
4 Precipitate or elute purified DNA from magnet
What is the purpose of protease K?
inactivates cellular DNases
How is DNA isolated?
by binding to a positively charged solid phase (DNA has a negative charge); usually consist of porous magnetic beads
Why are exogenous and endogenous RNases the biggest problem in RNA extraction?
They denature RNA
How do you keep RNases from denaturing RNA?
cells must be lysed quickly to allow denaturants to gain access and inactivate endogenous RNases
this is the technique used to amplify a target sequence of DNA
polymerase chain reaction (PCR)
What are the three steps of PCR?
1 denaturing the template DNA
2 annealing of primers
3 extending the strand
These three steps are one “cycle”
What are the components of the “Master Mix”?
deoxyribonucleotides (dATP, dCTP, dGTP, dTTP) often called dNTPs, DNA polymerase, buffer, Mg2+ (required by DNA polymerase), and primers
How is step #1 performed in PCR?
destroy H-bonds between strands (HEAT TO 95 degrees), GC bonds stronger than AT bonds and take more heat to destroy, some DNA has too many GC bonds and is too difficult to denature, avoid amplifying GC rich areas
How is step #2 performed in PCR?
decrease temperature to 50 degrees Celsius so primers can anneal
What makes a good primer?
should include at least 18 bases (usually 20-30); should have 50% GC and 50% AT, try not to put too many of the same bases in a row either; primer sequence should be unique to your target (amplifies your gene only)
Why can’t the 3’ ends of the primer pair be complementary?
May from primer Dimer when they anneal to each other or stem loop structures and will not amplify DNA
Primer pair melting points must be very similar by a difference of ______ or _____ which is better.
< 10 degrees C or < 5 degrees C is better
How is Step #3 performed in PCR?
Optimal temperature at 72’C; since human DNA denatures at this temp use DNA polymerase isolated from Thermus aquaticus bacteria (Taq polymerase); add master mix
What are the usual number of cycles in PCR?
25-35 cycles at 1-2min per cycle; more cycles=greater yield of product but also has a greater probability of generating artifacts
______ ________ is one of the methods that is used frequently to determine if there is amplification of the target.
Gel electrophoresis
This gel is used for larger DNA fragments in a horizontal box.
Agarose gel
This gel is used for smaller DNA fragments in a vertical box.
Polyacrylamide gel
DNA has a _______ charge due to phosphate groups.
Negative
DNA fragments move down the gel based on ______ .
Size
Describe the use of dyes to visualize DNA bands after electrophoresis.
Use ethidium bromide that fluoresces under UV light; can also use acridine orange or actinomycin D
How is autoradiography used to visualize DNA bands after electrophoresis?
Include radiolabeled dNTPs in master mix for PCR; overlay gel with photographic film to see bands.
Should the reagent control be positive or negative?
Negative! Master mix only! If positive it indicates contamination by PCR amplicons from previous runs
Should internal control be positive or negative?
Should be positive; involves amplifying another target and ensures there are no inhibitors that interfere with the amplification
Is the sensitivity control positive or negative?
Should be LOW positive
What happens when a tube filled with amplicons is opened?
DNA Amplicons move into the air which can contaminate the new PCR mixtures causing false positives
When primers are not specific enough what happens?
They bind at locations other than flanking the target so the wrong target is amplified
What happens when primers bind at more than one place?
Results in two amplified products
What happens when at lower temps a primer dissociates from the template and attaches to a different region?
A random product is amplified
This is a PCR reaction that goes for a set number of cycles and the amplification products must be detected/identified by an additional process upon completion of the PCR cycles.
Endpoint PCR
Endpoint PCR takes about _____ hours to complete but then requires more testing to be done to detect/identify the PCR products.
8 hours
This type of PCR detects and measures PCR products as they accumulate; involves use of probes with unique labels that fluoresce when they bind to the target sequence; fluorescence is measured for each cycle while cycling is in progress; occurs more quickly than endpoint PCR
Real Time PCR
Why is it desirable to detect RNA (not just DNA) through molecular testing?
Bacteria have a single circular piece of DNA but 10,000 copies of rRNA (larger target); viruses have either DNA or RNA
This is a PCR method that involves the use of the enzyme reverse transcriptase which makes DNA from RNA.
Reverse Transcriptase PCR
This is a method for direct detection of DNA or RNA sequences within cells and tissues without disrupting cells (no extraction or amplification); uses single stranded nucleic acid probes labeled with a fluorescent dye.
FISH
What are the three steps of FISH?
1 target cells or tissues are fixed on a slide
2 addition of labeled probe to hybridize to a complementary genomic DNA
3 detection of the probe via fluorescence microscopy
The probes in this method uses a peptide nucleic acid molecule (PNA) that mimics DNA instead of actual nucleic acid. The negatively charged sugar phosphate backbone of DNA is replaced with a non charged polyamide or peptide backbone. Primarily used for detecting RNA targets in situ.
PNA FISH
