Lab techniques used in biochemistry, for the USMLE exam
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:
- DNA is heated and denatured, this causes the separation of the strands.
- 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.
- DNA polymerase then replicates the desired DNA strands.
- 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.