Molecular Diagnostics

Question 1

What diagnostic technique consists of immobilizing target DNA on a solid support and then denaturing it, adding a probe, and base pairing them together?

Answer 1

Hybridization

Question 2

What is the purpose of hybridization?

Answer 2

Measures the degree of genetic similarity between DNA sequences and is used to determine the genetic distance between them (used extensively in phylogeny and taxonomy)

Question 3

With __________ blotting, both probe and target nucleic acid are DNA

Answer 3

Southern

Question 4

With __________ blotting, probe is ssDNA and target is mRNA

Answer 4

Northern

Question 5

What blotting technique is utilized to visualize target proteins?

Answer 5

Western

Question 6

Which molecular diagnostic technique is utilized in the early detection/confirmation of HIV based on circulating proteins in the patient’s bloodstream?

Answer 6

Western Blot

Question 7

Which blotting technique detects post-translational modifications on proteins?

Answer 7

Eastern

Question 8

Which molecular diagnostic technique determines which restriction fragments are associated with a gene?

Answer 8

Southern blotting

Question 9

Which molecular diagnostic technique is utilized to amplify isolated DNA regions?

Answer 9

PCR

Question 10

What is the clinical significance of PCR?

Answer 10

Earlier detection of microorganisms like HIV, bacterial, and fungal infections

Detection of specific genetic mutations like CF, thalassemia, hereditary hemochromatosis, Huntington’s, etc.

Question 11

What enzyme is utilized in PCR to synthesize the cloned DNA strand?

Answer 11

Taq polymerase - a heat stable polymerase that can withstand the thermocycler

Question 12

What is the primary advantage to PCR?

Answer 12

10^9 fold increase in DNA sequence in hours, from just a trace amount of sample

Question 13

What are the disadvantages to PCR?

Answer 13

Flanking sequence must be known for primer design

Taq polymerase is error prone b/c no 3–>5’ exonuclease activity

Amplification of contaminating DNA

Question 14

What type of PCR allows for real-time detection using sequence-specific fluorescent probes that show up at certain thresholds?

Answer 14

Real-time PCR (qPCR)

Question 15

What molecular diagnostic technique is primarily used in forensics, paternity testing, prenatal diagnosis (amniocentesis), IEM screening, and genetic carriers?

Answer 15

Restriction Fragment Length Polymorphism (RFLP) and VNTR

Question 16

With RFLP, DNA samples are extracted from evidence and individuals are cleaved by _______ enzymes, resolved by _____________, then analyzed by _________ blotting

Hybridization of the blot to radiolabled DNA followed by autoradiography reveals specific DNA sequence

Answer 16

Restriction
Electrophoresis
Southern

Question 17

______ is useful in identifying and determining the severity of inherited diseases like Huntingtons, Fragile X, and Freidrich Ataxia when used in conjunction with PCR and southern blotting

Answer 17

VNTR

Question 18

Large scale production of ____________ _________ is used to create insulin, growth hormone, clotting factors, and HepB surface antigens (vaccine)

_______ of the therapeutic protein is inserted into expression vectors (plasmids), then engineered to allow high levels of replication, transcription, and translation, allowing large scale production and rapid purification

Answer 18

Recombinant proteins

cDNA

Question 19

Recombinant DNA is produced by _______ DNA together from 2 sources. It can replicate and be expressed into proteins in microorganisms

Answer 19

splicing

Question 20

What are the methods of generating recombinant DNA?

Answer 20

Restriction endonucleases are chosen to target specific gene to cut DNA and produce restriction fragments, which are then cloned

Cloned fragements are re-isolated from recombinant vectors and sequenced

Question 21

What molecular technique is used for analyzing and altering genes and gene products, providing material for genetic analysis, and providing sources of gene products like proteins and insulin?

A. VNTR
B. Recombinant DNA
C. PCR
D. Hybridization

Answer 21

B. Recombinant DNA

Question 22

How can recombinant DNA technology be used to improve insulin?

Answer 22

Human insulin has Pro-28 and Lys-29; by switching these two, Lispro is faster acting, more readily absorbed, and maintains tighter glycemic control when used with insulin

Insulin Aspart = proline –> aspartic acid

Insulin Glargine = Asparagine –> glycine, adds 2 arginine

Question 23

How can recombinant DNA technology be used to treat cancer, rheumatoid arthritis, Crohn’s disease, Hepatitis, and more?

Answer 23

Production of monoclonal antibodies

Question 24

What molecular diagnostic technique tests for antigen-antibody reactivity with a color change indicating a positive test?

Answer 24

ELISA

Question 25

What is the difference between an indirect ELISA and a sandwich ELISA?

Answer 25

Indirect - detects antibody in a sample

Sandwich - detecs antigen in a sample

Question 26

What molecular diagnostic technique is utilized in MI detection?

Answer 26

Sandwich ELISA

Troponin proteins act as the antigens. Horseradish peroxidase (HRP) is the antibody.

Question 27

What molecular diagnostic technique is utilized in the diagnosis of HIV? How are false-positives or false-negatives confirmed?

Answer 27

Hybrid sandwich/indirect ELISA [immobilized HIV antigen is used to capture human Abs, then reacted with IgG]

Confirmation by Western blotting

Question 28

What molecular diagnostic technique is utilized in at-home pregnancy testing?

Answer 28

Sandwich ELISA

[test has monoclonal Abs that recognize hCG]