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USMLE Biochemistry > Laboratory techniques > Flashcards

Laboratory techniques Flashcards

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USMLE® Step 1 flashcards
1
Q

What are the uses of PCR?

A
  • Make more DNA
  • Determine if DNA is present
  • Determine amount of DNA (how quickly does it amplify)
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2
Q

What are the ingredients in PCR?

A
  • Sample (DNA)
  • DNA polymerase
  • Primer
  • NTs
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3
Q

Describe one PCR cycle?

A

Heat sample
- DNA denatures into SS

Cool sample
- Primer anneals (binds) complementary DNA

Warm sample
- DNA polymerase elongates from primer

Repeat

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4
Q

What is the purpose of adding fluorescent dye to PCR?

A

Means you can quantify how much DNA is in a sample based on how quickly the DNA becomes florescent

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5
Q

How can PCR be useful in HIV?

A

cDNA is amplified

- Amount of cDNA produced over time will indicate VIRAL LOAD

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6
Q

What else is required in HIV PCR?

A

Reverse transcriptase to make cDNA

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7
Q

What can PCR be used to diagnose?

A

Herpes simplex encephalitis

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8
Q

What different blots identify what molecular structres?

A
  • Southern blot: DNA
  • NoRthern blot: RNA
  • Western blot: Proteins

West produces proteins
South America has mixed DNA, Edward Southern invented it

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9
Q

What identifies DNA in a sample in the Northern blot?

A

Probe

  • Single stranded DNA molecule, carries radioactive or chemical markers
  • Once bound the markers reveal DNA sample
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10
Q

What are the steps involved in the southern blot technique?

A
  • DNA sample enzymatically cleaved by restriction nucleases
  • Pieces put on gel electrophoresis, seperated by size
  • Blotting then occurs, filter paper put over
  • Probe added, unbound probe washed away, leaving only bound probe, filter paper exposed to film which will reveal the bound DNA
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11
Q

What is RFLP (Restriction fragment length polymorphisms) analysis?
Describe its purpose and what the technique involves?

A

Technique used to characterise genes

  • Restriction endonucleases used
  • Analysis of fragments can reveal what genes are present
  • Different genes have different lengths
  • Southern blotting then used to detect lengths after fragmentation
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12
Q

What do Restriction endonucleases do?

A

DNA cutting enzymes - cut DNA at specific base sequences

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13
Q

Restriction fragment lenght piolymorphism technique is useful in what?

A

Working out inheritance of different alleles

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14
Q

What disease can be diagnosed through RFLP (Restriction fragment length polymorphisms) analysis?

A

Sickle cell

- Different Beta globin genes will have different lengths

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15
Q

Northern blot is useful for assessing what?

A

mRNA levels (gene expression)

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16
Q

What is used instead of a probe in Western blot technique?

A

Specific antibody to protein

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17
Q

What diseases may be detected through antibodies with Western blot?

A
  • IgG or IgM in Lyme disease

- IgG HIV-1

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18
Q

What is the SouthWestern blot used to study?

A

DNA-protein interactions

- Especially transcription factors

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19
Q

Describe the SW blot

A

Proteins seperated by electrophoresis

- DNA then added as a probe - to see if they interact

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20
Q

What is flow cytometetry?

A

Analysis of cells as they flow through a liquid through a narrow steam
- Used to analyse cells by size and surface proteins

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21
Q

What are the key components of a flow cytometer?

A
  • Flow cell: moves cells through machine
  • Laser: light scattered by cells
  • Photodetector: detects light scatter
22
Q

What will the forward scatter in flow cytometetry detect?

A

Size of cell

23
Q

What will the side scatter in flow cytometetry detect?

A

Granularity

24
Q

What is antibody staining with flow cytometery useful for?

A

Presence of preotein in cells

25
Q

What are the specific antibodies tagged with in antibody staining?

A

Unique fluorochrome

- Flow cytometer will detect fluorochrome if bound to cell

26
Q

How can flow cytometry be used to detect fetal maternal hemorrhage?

A

Monoclonal antibody to hemoglobin F

- Detects fetal hemoglobin in red cells of the maternal circulation

27
Q

What do fluorescently-labeled monoclonal antibodies bind to on RBCs?

A

GPI (glycosylphosphatidylinosito) anchored proteins

28
Q

What disease will show reduced or absent RBCs after flow cytometry?

A

Paroxysmal nocturnal hemoglobinuria

- This is because they do not contain GPI anchored proteins which is what antiBs bind to

29
Q

What are some examples on GPI anchored protiens?

A
  • Decay accelarating facgtor (DAF/CD55)

- MAC inhibitory protein (CD59)

30
Q

What does ELISA (Enzyme-linked immunosorbent assay test detect?

A

Antigens and antibodies in the serum

31
Q

What are the different forms of ELISA test?

A
  • Direct
  • Indirect
  • Sanwich
  • Competitive
32
Q

What are the steps involved in the direct ELISA test?

A

Add serum to plate -> antigen secured to surface

  • Wash away liquid
  • Enzyme-labeled antibody specific to antigen added
  • Wash away unbound antibodies
  • Substrate added -> colour change
  • Enzyme-linked antibodies directly bind to the antigen
33
Q

What is the indirect ELISA test involve?

A

Serum added for analysis

  • Antibody added and binds to antigen
  • Antibody and not enzyme linked
  • Add an enzyme-labeled secondary antibody
  • Substrate added -> colour change -> identification of antigen
34
Q

What type of ELISA test requires a second antibody?

A

Indirect ELISA

- Secondary antibody is enzyme-linked - which will show colour change

35
Q

What is more commonly done the direct or indirect ELISA test?

A

Indirect

36
Q

What type of ELISA test requires a capture antibody first which the antigen then binds to?

A

Sandwich ELISA

  • Antigen sandwiched between the 2 antibodies
  • Can be direct or antigen
37
Q

What are the advantages of the sandwich ELISA test?

A
  • High specificity
  • Works with complex samples (antigen does not require purification)
  • Can use secondary antibody like indirect
38
Q

What is the competitive ELISA test?

A

Primary antibody incubated with the sample
- Antigen-antibody complexes form
- These are washed away
- Secondary antiB and substrate will be added
- More antigen = more binding = less free antibody
-

39
Q

What does a bigger colour change indicate in competetive ELISA test?

A

LESS antigen in sample

40
Q

What can ELISA be used to detect?

A

HIV antibody (indirect)

41
Q

What antigen can be detected to identify HIV on ELISA?

A

HIV p-24 antigen

- Often sandwich ELISA used

42
Q

What can a DNA Microarray (chip or microchip) contain?

A

Glass, plastic or silica

43
Q

What does DNA MIcroarray involve the use of to identify a sample?

A

Thousands of probes

  • Used to test a sample of DNA with fluorescent markers
  • Sample hybridizes with complementary bases
  • Computer detects which probes bind sample
44
Q

How can DNA Microarray be used to identify gene expression?

A

Cellular mRNA can be collected and converted to cDNA

  • cDNA can be tested using microarray
  • Can then determine which genes have been expressed
45
Q

Why may we want to identify gene expression? (using DNA microarray)
Give an example

A

Cancer

  • Which genes are active vs inactive
  • Cancer cells vs normal cells
46
Q

What is copy number variation and when can it be useful?

A

Some cells contain incr/decr copies of genes DNA

- Increased/decr copies linked to disease and can be identified via an increased or decreased fluoresence

47
Q

What are SNPs?

A

Single Nucleotide polymorphisms

  • Genes with variations in a single nucleotide
  • Variations represented in microarray
  • Binding indicates which SNP present in sample gene
  • Many SNPs associated with disease and also preserved within families
48
Q 
Describe FISH (Fluorescent in situ hybridisation)
What can it identify
A
  • Fluorescent DNA probe binds to specific gene site
  • Localises genes to a chromosome
  • Can determine which chromosome contains a gene
49
Q

What is hybridization?

A

Binding of probe to complimentary DNA/RNA

50
Q

What part of the cell cycle is FISH mostly done in?

A

Metaphase

- Cells arrested in mitosis so chromosomes are visible individually

51
Q

What can FISH identify?

A

Chromosomal abnormalities (test cells compared to normal cells)

  • Microdeletions as no fluoresence on chromosome (DiGeorge)
  • Translocation
  • Duplication (extra site of fluorescence)

USMLE Biochemistry (44 decks)

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