Molecular Diagnostic methods Part B

Question 0

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

Answer 0

primer

Question 1

artificial replication; important when the nucleic acid of interest in a clinical sample is very small (viral and bacterial infections)

Answer 1

amplification

Question 2

a short single-stranded segment of nucleic acid

Answer 2

oligonucleotide

Question 3

In vitro molecular diagnostic techniques function in detecting particular sequences in _____ ______

Answer 3

nucleic acids

Question 4

If you use anticoagulated whole blood in testing it cannot contain what?

Answer 4

heparin

Question 5

List the steps in a typical DNA extraction process (4)

Answer 5

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

Question 6

What is the purpose of protease K?

Answer 6

inactivates cellular DNases

Question 7

How is DNA isolated?

Answer 7

by binding to a positively charged solid phase (DNA has a negative charge); usually consist of porous magnetic beads

Question 8

Why are exogenous and endogenous RNases the biggest problem in RNA extraction?

Answer 8

They denature RNA

Question 9

How do you keep RNases from denaturing RNA?

Answer 9

cells must be lysed quickly to allow denaturants to gain access and inactivate endogenous RNases

Question 10

this is the technique used to amplify a target sequence of DNA

Answer 10

polymerase chain reaction (PCR)

Question 11

What are the three steps of PCR?

Answer 11

1 denaturing the template DNA
2 annealing of primers
3 extending the strand
These three steps are one “cycle”

Question 12

What are the components of the “Master Mix”?

Answer 12

deoxyribonucleotides (dATP, dCTP, dGTP, dTTP) often called dNTPs, DNA polymerase, buffer, Mg2+ (required by DNA polymerase), and primers

Question 13

How is step #1 performed in PCR?

Answer 13

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

Question 14

How is step #2 performed in PCR?

Answer 14

decrease temperature to 50 degrees Celsius so primers can anneal

Question 15

What makes a good primer?

Answer 15

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)

Question 16

Why can’t the 3’ ends of the primer pair be complementary?

Answer 16

May from primer Dimer when they anneal to each other or stem loop structures and will not amplify DNA

Question 17

Primer pair melting points must be very similar by a difference of ______ or _____ which is better.

Answer 17

< 10 degrees C or < 5 degrees C is better

Question 18

How is Step #3 performed in PCR?

Answer 18

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

Question 19

What are the usual number of cycles in PCR?

Answer 19

25-35 cycles at 1-2min per cycle; more cycles=greater yield of product but also has a greater probability of generating artifacts

Question 20

______ ________ is one of the methods that is used frequently to determine if there is amplification of the target.

Answer 20

Gel electrophoresis

Question 21

This gel is used for larger DNA fragments in a horizontal box.

Answer 21

Agarose gel

Question 22

This gel is used for smaller DNA fragments in a vertical box.

Answer 22

Polyacrylamide gel

Question 23

DNA has a _______ charge due to phosphate groups.

Answer 23

Negative

Question 24

DNA fragments move down the gel based on ______ .

Answer 24

Size

Question 25

Describe the use of dyes to visualize DNA bands after electrophoresis.

Answer 25

Use ethidium bromide that fluoresces under UV light; can also use acridine orange or actinomycin D

Question 26

How is autoradiography used to visualize DNA bands after electrophoresis?

Answer 26

Include radiolabeled dNTPs in master mix for PCR; overlay gel with photographic film to see bands.

Question 27

Should the reagent control be positive or negative?

Answer 27

Negative! Master mix only! If positive it indicates contamination by PCR amplicons from previous runs

Question 28

Should internal control be positive or negative?

Answer 28

Should be positive; involves amplifying another target and ensures there are no inhibitors that interfere with the amplification

Question 29

Is the sensitivity control positive or negative?

Answer 29

Should be LOW positive

Question 30

What happens when a tube filled with amplicons is opened?

Answer 30

DNA Amplicons move into the air which can contaminate the new PCR mixtures causing false positives

Question 31

When primers are not specific enough what happens?

Answer 31

They bind at locations other than flanking the target so the wrong target is amplified

Question 32

What happens when primers bind at more than one place?

Answer 32

Results in two amplified products

Question 33

What happens when at lower temps a primer dissociates from the template and attaches to a different region?

Answer 33

A random product is amplified

Question 34

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.

Answer 34

Endpoint PCR

Question 35

Endpoint PCR takes about _____ hours to complete but then requires more testing to be done to detect/identify the PCR products.

Answer 35

8 hours

Question 36

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

Answer 36

Real Time PCR

Question 37

Why is it desirable to detect RNA (not just DNA) through molecular testing?

Answer 37

Bacteria have a single circular piece of DNA but 10,000 copies of rRNA (larger target); viruses have either DNA or RNA

Question 38

This is a PCR method that involves the use of the enzyme reverse transcriptase which makes DNA from RNA.

Answer 38

Reverse Transcriptase PCR

Question 39

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.

Answer 39

FISH

Question 40

What are the three steps of FISH?

Answer 40

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

Question 41

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.

Answer 41

PNA FISH