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Flashcards in Biochem - Lab Techniques Deck (47)
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1

What is the purpose of using DNA polymerase chain reaction?

To amplify the number of copies of DNA fragments

2

How is the size of a DNA fragment determined from an agarose gel?

A DNA ladder of known lengths is run alongside the polymerase chain reaction fragments

3

How is DNA separated into two strands during polymerase chain reaction?

By heating

4

In polymerase chain reaction, premade _____ are used to anneal to specific DNA sequences that will then be amplified.

Primers

5

What enzyme is used in polymerase chain reaction to amplify DNA?

Heat-stable DNA polymerase

6

What are the three steps of DNA polymerase chain reaction in order?

Denaturation, annealing, elongation

7

What is the advantage of using a heat-stable DNA polymerase?

The polymerase is not denatured by heating and can be reused for multiple cycles of polymerase chain reaction

8

Following PCR, how can products be separated and analyzed?

Agarose gel electrophoresis

9

Which would travel further on an agarose gel: a 10-kD or an 100-kD DNA fragment?

The 10-kD fragment; smaller molecules travel further

10

Describe the technique of a Western blot.

Sample protein is separated via gel electrophoresis and transferred to a filter; labeled antibody is used to bind the protein of interest

11

What blot technique detects specific sequences of DNA?

Southern blot

12

How does the mnemonic "SNoW DRoP" help one remember which type of blot is used to detect DNA sequences, RNA sequences, or proteins?

Southern = DNA, Northern = RNA, Western = Protein

13

Describe the technique of a Southern blot.

DNA is run on an electrophoresis gel and transferred to a filter; the DNA on the filter is denatured and exposed to a labeled DNA probe, and then the double-stranded DNA is visualized when the filter is exposed to film

14

Describe the technique of a Northern blot.

RNA is run on an electrophoresis gel and transferred to a filter; the RNA on the filter is exposed to a labeled DNA probe, and then the hybrid DNA-RNA molecule is visualized when the filter is exposed to film

15

Which assay would allow you to detect single nucleotide polymorphisms?

Microarrays

16

How can a microarray profile gene expression levels?

The microarray scanner can detect relative amounts of complementary binding to probes

17

Describe the technique of using a microarray to detect gene expression levels.

Thousands of nucleic acid sequences are arranged in grids on glass or silicon. DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amounts of complementary binding

18

True or False? In the microarray biology technique, DNA or RNA probes are hybridized to the chip.

True; either DNA or RNA probes may be used

19

True or False? Enzyme-linked immunosorbent assays test antigen-antibody reactivity.

True

20

What information would an enzyme-linked immunosorbent assay using a test antibody coupled to a color-generating enzyme give you?

It would tell you whether a certain antigen is present in the patient's blood

21

Describe the difference between enzyme-linked immunosorbent assay techniques used to detect antigens and antibodies.

A test antigen labeled with a color-generating enzyme can be used to determine the presence of a specific antibody; a test antibody labeled with a color-generating enzyme can be used to see if a certain antigen is present

22

Describe the technique of an enzyme-linked immunosorbent assay

A test antigen that is labeled with a color-generating enzyme can be used to determine the presence of a specific antibody; if the antibody is present, the solution will have an intense color reaction

23

True or False? The sensitivity and specificity of the enzyme-linked immunosorbent assay approaches 100%.

True; however, false-negative and false-positive results may still occur

24

What is the advantage of a fluorescence in situ hybridization analysis over a karyotype?

It allows researchers to identify anomalies at a molecular level, including deletions that are too small to see on a karyotype

25

What allows fluorescence in situ hybridization analysis to detect deletions too small to visualize on karyotype?

The fluorescent probes used can bind small segments of DNA and illustrate the presence or absence of genetic material

26

What laboratory technique is represented with the acronym FISH??

Fluorescent In Situ Hybridization

27

What does fluorescent in situ hybridization allow researchers to do?

Directly visualize the location of a certain protein or gene on a molecular level through a fluorescent probe that binds to a site of interest

28

In cloning, what is the significance of inserting DNA to be replicated into plasmids also containing antibiotic-resistance genes and then growing the bacteria on media containing the antibiotic that bacteria is resistant to?

This technique selects for replication of the plasmid containing genes for antibiotic resistance AND the DNA of interest

29

What is the role of restriction enzymes in DNA cloning?

Restriction enzymes cleave DNA at 4-6 base pair palindromic sequences, allowing for insertion of a fragment into a plasmid

30

What are the three steps of DNA cloning in order?

Insertion of DNA fragments into bacterial plasmids, cleavage of DNA allowing for insertion, isolation of mRNA to be exposed to reverse transcriptase