Advances in Nucleic Acid Technology

Question 1

DNA polymorphism is used to

Answer 1

determine what/whom is the source of infection, NOT identify if the agent is present

Question 2

Gel electrophoresis to compare plasmid sizes between 2 bacterium isolated from separate patients can tell you

Answer 2

whether or not the bacteria are from the same source

Question 3

How would one perform a gel electrophoresis?

Answer 3

isolate and purify plasmids, run gel to separate plasmid DNA by size, image bands

Question 4

Positive match on gel electrophoresis

Answer 4

same number and size of plasmids

Question 5

Restriction Fragment Length Polymorphism (RFLP)

Answer 5

DNA chromosome fingerprinting based on restriction sites, common restriction sites will yield fragments of the same length

Question 6

How would one perform a RFLP?

Answer 6

isolate bacterial chromosome and purify, treat with restriction endonucleases, separate based on size of fragments, detect with Southern or Northern blots

Question 7

Positive match on Southern blotting for RFLP or ribtoyping

Answer 7

same number and size of DNA fragments

Question 8

Pulse Field Gel Electrophoresis

Answer 8

isolate chromosomal DNA by gently LYSING open the cell, treat with restriction endonucleases, produce large DNA fragments, separate and image

Question 9

DNA polymorphisms studies are relevant because

Answer 9

they differentiate b/w strains and determine if there was a common source responsible (epidemiological purposes)

Question 10

Hybridization reaction

Answer 10

must have a known probe sequence that you are checking for, check to see if probe hybridizes with ssDNA

Question 11

Southern blotting

Answer 11

DNA (separate fragments, incubate with ssProbe)

Question 12

Northern blotting

Answer 12

RNA (separate fragments, incubate with ssProbe)

Question 13

Colony blotting

Answer 13

bacteria grown on a mesh are lysed, hybridization reaction occurs if DNA matches ssProbe

Question 14

Use of Southern or Northern blotting is clinically important for

Answer 14

diagnosing disease, detection of viral NA, identifying the infectious agent

Question 15

2 types of amplification

Answer 15

signal amplification and target amplification

Question 16

Signal Amplification

Answer 16

multiple markers binding 1 target for “signal amplification”, this is cheaper than amplifying the target sequence

Question 17

Amplification of Target sequence via PCR

Answer 17

must have known ssProbe, heat-stable DNA polymerase, and nucleotides

Question 18

3-step process of PCR

Answer 18

denature, anneal primers, extension of primers

Question 19

PCR is used to amplify small amounts of NA in order to

Answer 19

screen blood, detect virus or bacteria, detect genetic defect of neoplasm marker

Question 20

Other techniques to amplify target genome

Answer 20

Ligase chain reaction LCR, transcription-mediated amplification (TMA)

Question 21

Why use LCR or TMA over PCR?

Answer 21

Cheaper, do not use heat-stable DNA pol