Molecular Diagnostics

Question 1

What is molecular diagnostics used for?

Answer 1

Detecting infectious agents and diagnose inherited disorders- inorder for this to happen the genome must be known

Question 2

Hybridization method

Answer 2

Single stranded DNA binds to a complementary strand of DNA or RNA to detect or quantify a section of DNA/RNA in a sample.

Question 3

How is the hybridization method done?

Answer 3

Target DNA is converted to single stranded DNA then immobilized on a solid support. Single stranded oligonucleotides are added with a marker to the gel.

Question 4

What are the four types of blotting techniques?

Answer 4

Southern- DNA to DNA
Northern- DNA to RNA
Western- Protein
Eastern- PTM

Question 5

What is PCR?

Answer 5

Polymerase chain reaction- a sample of DNA can be amplified

Question 6

How does PCR work?

Answer 6

Double stranded DNA (sample) is subjected to high temp and denatured to single stranded DNA. Primers designed to be compliment DNA flank each end and allowed to anneal. Taq polymerase and dNTPs are added to synthesis new DNA. This is repeated multiple times and DNA sample is amplified.

Question 7

Disadvantages to PCR?

Answer 7

Need to know the sequence of flanking DNA, error prone and contaminating DNA will be amplified as well

Question 8

Advantages to PCR?

Answer 8

Only a small sample is needed for amplification

Question 9

What is qPCR?

Answer 9

Quantitative PCR allows for real time quantification of copy number in a sample. This is done by adding a fluorescent marker to the oligotide that only fluoresces in the presence of PCR product.

Question 10

What is the advantage of qPCR technique?

Answer 10

Detects levels of an infectious agent
Determine levels of gene expression
EXAMPLE- Can compare gene expression levels in varying cell types by determining how many cycles it takes to reach a certain amount

Question 11

What is RFLP?

Answer 11

Restriction Fragment Length Polymorphism- Since genomes differ by 1 in every 1000 basepairs restriction enzymes can be used to cleave DNA and individuals will produce different patterns when cleaved DNA applied to a gel.

Question 12

Using RFLP for sickle cell detection

Answer 12

Normal Beta globulin has 2 DDEL restriction sites while mutated globulin has 3 DDEL- when added to a gel sickle cell identification can be made if the gel only contains one band.

Question 13

What is VNTR?

Answer 13

Variable number of tandem repeats- short tandem repeats of DNA occur in individuals by in vary in number. These sections can be isolated by restriction enzymes or PCR. Can identify disease or determine severity

Question 14

Recombinant proteins

Answer 14

cDNA of a protein inserted into a vector then allowed to replicate. Allows for large scale replication and isolation

Question 15

How are antibodies produced?

Answer 15

An antigen is inserted into a subject and antibodies are produced. These antibodies are then isolated and added to tumor cells that allow for immortalization

Question 16

What is an ELISA?

Answer 16

Enzyme-linked Immunosorbent Assay- Test for levels of antigen or antibody in a sample- a reactive enzyme is added to the assay to enduce color change that can quantify a sample.

Question 17

What is an indirect ELISA?

Answer 17

Detecting levels of antibody in a sample. An antigen is immobilized on a substrate and the sample is passed through capturing antibodies.

Question 18

What is a sandwhich ELISA?

Answer 18

Detecting levels of an antigen in a sample. An antibody is immobilized on a substrate and the sample is passed through, capturing antigen.