Lab techniques used in biochemistry, for the USMLE exam

by / Monday, 30 August 2010 / Published in Biochemistry

The Polymerase Chain Reaction (PCR)

This technique is used when a large number of DNA copies are needed.  The steps to creating multiple copies of DNA fragments through the PCR are as follows:

  1. DNA is heated and denatured, this causes the separation of the strands.
  2. The denatured DNA is cooled, and DNA primers are added to the mix, these adhere to each individual strand of DNA at the location that will be amplified.
  3. DNA polymerase then replicates the desired DNA strands.
  4. This process is repeated until the desired number of DNA is achieved.

ELISA (Enzyme-Linked Immunoabsorbent Assay)

This technique is used as a means of detecting the presence of an antibody or an antigen in a sample.  The antibody or antigen that is added is linked to an enzyme, then a test solution is added to see if an intense color illuminates, indicating that there is a positive result.

–       This test is most commonly used when looking for HIV.

–       Sensitivity and specificity for the ELISA are extremely high, both approaching 100%, however they are not perfect, and false results do occur.

SOUTHERN BLOT TECHNIQUE

This technique is used to detect specific sequences of DNA.  The technique combines the transfer of electrophoresis-separated DNA fragments and membrane filtration, and then fragments are detected by probe hybridization.

WESTERN BLOT TECHNIQUE

This is a technique used to detect specific proteins, separating native or denatured proteins by the length of the polypeptide.  These proteins are then transferred to a membrane where they are probed using antibodies specific to the target protein.

NORTHERN BLOT TECHNIQUE

Is a technique used to study gene expression by RNA detection in a sample.  This technique allows for the detection of cellular control by determination of gene expression levels during differentiation and morphogenesis.

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