USMLE annoying lab techniques

Just covering the bases of some of the super-annoying lab techniques that are known to show up on the Step 1.

I wanted to pass out at the idea of writing this post, so thankfully I have a battery-acid coffee to get me through this one.

Southern blot

• DNA is run on a gel, transferred to a membrane, and visualized with a complimentary probe
• Good for paternity testing

Northern blot

• Same as southern blot but with RNA, not DNA.

Western blot

• Same technique, but with protein, not nucleic acid.
• The protein is visualized using a complimentary antibody.
• This is the answer whenever they ask for the confirmatory test for HIV (p24 antigen testing, a newer test, can also be done, but Western blot is the gold standard confirmatory test)
• It is also the answer when they ask for how to confirm Lyme disease (serologies are also used, but the USMLE tests Western blot for Lyme).

Western blot is almost always the answer to “what is the confirmatory test?” For HIV, ELISA is the sensitive screening test (↓ false-negatives); Western blot is the specific confirmatory test (↓ false-positives). Either PCR or HIV culture is used in neonates.

Southwestern blot

• A type of Western blot
• A protein is run on a gel and then visualized with an oligonucleotide probe.
• This is great for identifying DNA-binding proteins.

Restriction fragment length polymorphism (RFLP)

• Restriction enzymes are used to digest DNA
• The DNA fragments are then separated according to length
• Although a relatively obsolete technique, on the Step1, it has been known to show up as another way to do paternity testing.

DNA fingerprinting

• A DNA technique that is used for paternity testing and forensics
• Looks at variable number tandem repeats (VNTRs), which are short DNA sequences that differ in number of repeats among individuals.

On the USMLE, Southern blotting, RFLP and DNA fingerprinting are all techniques used for paternity testing. Thrilling.

DNA microarray

• A chip hybridized with RNA and/or DNA probes is used to simultaneously detect expression of thousands of genes
• For the Step 1, the only thing you need to know about this technique is that it is able to detect single nucleotide polymorphisms (SNPs).

Sequencing

• Uses dideoxynucleotides to randomly terminate growing strands of DNA, then the DNA is separated on a gel and read by the order of the bands on the gel.
• Just know this description refers to sequencing.

Polymerase chain reaction (PCR)

• Used to amplify a DNA fragment in order to make many copies
• The two DNA strands are separated at high temperature
• They are then amplified using a heat-stable DNA polymerase and two different primers (one for each strand) that anneal to the separated strands at lower temperature.

You might be given a dsDNA sequence asked to pick the primers.

If you are given, e.g.:

3’ ATGACTAGCTAGA 5’ = non-coding strand

5’ TACTGATCGATCT 3’ = coding strand

Bear in mind that DNA synthesis always occurs 5’→3’ and you need a primer that can amplify each strand.

One of the primers will be complimentary to the 3’-end of the coding strand; the other primer will be identical to the 5’-end of the coding strand.

Therefore, the primers would be:

5’ TACTGA 3’ (identical to 5’-end of coding strand; amplifies the 3’-end of the non-coding strand in the 3’→5’ direction and is synthesized 5’→3’), and

5’ AGATCG 3’ (complimentary to 3’-end of coding strand; amplifies the 3’-end of the coding strand in the 3’→5’ direction and is synthesized 5’→3’).

Reverse transcription PCR is used to detect mRNA levels. The mRNA is first converted to cDNA, then PCR is done. When determining genotype (e.g., of an embryo, when both parents are CF carriers), isolate DNA, run PCR first, then do a Southern blot.

Enzyme-linked immunosorbent assay (ELISA)

• Sensitive test (↓ false-negatives) and is commonly used as a screening test
• As mentioned above, the USMLE is obsessed with you knowing that ELISA is the main screening test for HIV
• Two main types of ELISA tested on Step 1: direct and indirect
  • Direct ELISA = you are trying to detect antigen in the patient’s serum
  • Indirect ELISA =you are trying to detect antibody in the patient’s serum 

Direct ELISA

• Coat a well with known antibody.
• Add patient’s serum. If the antigen is present in the serum, it will bind the antibody on the well.
• So now there is a well with an Ab-Ag complex bound to the well.
• Now add secondary antibody (Ab₂).
• This secondary antibody (anti-human immunoglobulin) is bound to a horseradish peroxidase enzyme (Ab₂-E) that is capable of oxidizing a chromogen (substrate that produces color when oxidized).
• So now there’s a well with Ab-Ag-Ab₂-E bound.
• Then the chromogen is added.
• The enzyme on the Ab₂ oxidizes it and color is generated.
• This color is measured using an absorbance reader.

So for direct ELISA, since you are trying to detect antigen in the patient’s serum, you need the plate to initially be coated with known antibody. This also means that the secondary antibody binds the patient’s antigen.

Indirect ELISA

• Coat a well with known antigen.
• Add patient’s serum. If the antibody is present in the serum, it will bind the antigen on the well.
• So now there is a well with an Ag-Ab complex bound to it.
• Then add secondary antibody (Ab₂). For indirect ELISA, this is called anti-Human immunoglobulin.
• This secondary antibody (anti-human immunoglobulin) is bound to a horseradish peroxidase enzyme (Ab₂-E) that is capable of oxidizing a chromogen (substrate that produces color when oxidized).
• So now there’s a well with Ag-Ab-Ab₂-E bound.
• Then the chromogen is added.
• The enzyme on the Ab₂ oxidizes it and color is generated.
• This color is measured using an absorbance reader.

So for indirect ELISA, since you are trying to detect antibody in the patient’s serum, you need the plate to initially be coated with known antigen. This also means that the secondary antibody binds the patient’s antibody.

• In indirect ELISA, the secondary antibody binds the Fc region of the primary antibody because the primary antibody’s Fab regions are bound to the antigen coating the well.

For either direct or indirect ELISA, if they ask you for the name of the enzyme that is coupled to the Ab2, the answer is horseradish peroxidase.

For indirect ELISA, if they ask you for the name of the secondary antibody that binds the patient’s antibody, the answer is anti-human immunoglobulin.

An antibody monomer has one Fc region and two Fab fragments. Papain cleaves antibodies into one Fc region and two separate Fab fragments. → (1 Fc + 2 Fab) Pepsin cleaves Abs into one Fc region and two connected Fab fragments. → (1 Fc + 1 (Fab)2) Just remember that Pepsin chops the whole Ab in half; Papain doesn’t.

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