DNA Diagnostic Laboratory at Johns Hopkins  
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General Test Information
Reason for Genetic Testing
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Technical Information
Sequencing:  Sequencing looks at each DNA base contained within the areas of the gene that code for the protein and a limited number of nucleotides around each splice site. DNA is amplified via polymerase chain reaction and sequencing is generally performed in both the forward and reverse directions.  Sequencing works through comparing the patient nucleotides to those in a reference sequence and looking for any differences.  Sequencing is designed to detect changes to single nucleotides, although it will also detect small scale deletions or duplications. 
MLPA Multiplex Ligation Probe Amplification (MLPA) assesses gene deletions and/or duplications using several paired probes.  If the DNA hybridizing to the probes is present, the probes are ligated together.  If a deletion is present, the paired probes will not ligate.  MLPA works through measuring the concentration of ligated probe pairs and comparing the concentration with known negative and positive controls. 
Test Limitations
  Assessing the significance of identified mutations may require clinical evaluation and/or genetic testing of family members.  All tests may produce false negative and false positive results (see the analytic and clinical sensitivity for each test).  Below are some additional test limitations that are dependent on the technical platform used for the test. 
Sequencing:   The sequencing test may not analyze both copies of the gene:  Polymorphisms under the primers used for PCR may prohibit amplification.  One allele may also be preferentially amplified during PCR.  

The sequencing assay looks only at the coding regions of the gene:  either all of the coding regions or a select set of them, as explained on each test page.  The sequencing assay does not typically include non-coding intronic sequences, promoter regions or 3"UTR unless stated on the test page.  Disease-causing mutations can be located in these regions, and a sequencing assay will miss them.

Sequencing cannot detect large-scale deletions or duplications (beyond several nucleotides).

Sequencing will not detect mosaicism for a sequence variation.

MLPA: MLPA assesses discrete areas of the gene.  Deletions that fall between probes will not be identified.

MLPA will not detect rearrangements of DNA that may affect gene function but do not disturb the overall copy number as assessed by the individual probes.