MOLECULAR TESTING

• Specimen


  • Vic (WT)
  • Fam (MU)
  • Ratio (MU/WT)
  • Average
• Negative control


  • 2.973
  • 0.804
  • 0.270
  • 2.986
  • 0.756
  • 0.253
  • 0.262
• Positive control


  • 1.073
  • 3.395
  • 3.130
  • 1.109
  • 3.359
  • 3.061
  • 3.096
• No template control


  • 0.066
  • 0.026
  • 0.394
  • 0.069
  • 0.027
  • 0.391
• Sensitivity control


  • 2.999
  • 1.074
  • 0.358
  • 2.989
  • 1.077
  • 0.359
  • 0.359
• Patient


  • 3.06
  • 2.695
  • 0.881
  • 3.008
  • 2.75
  • 0.914
  • 0.898
• Figure 29.1, cont'd (C), Table of the fluorescent values after 35 cycles of PCR showing the relative ratio of FAM/VIC fluorescence for each of the specimens. Specimens with a FAM/VIC ratio greater than the sensitivity or referent control are considered mutant.
• Molecular biology techniques enhance the diagnostic team's ability to predict or identify an increasing number of diseases in the clinical laboratory. Molecular techniques also enable clinicians to monitor disease progression during treatment, make accurate prognoses, and predict the response to therapeutics.
• The short interval required to perform molecular diagnostic tests and analyze their results is an additional positive aspect of this type of testing, resulting in more efficient patient management, especially in cases of infection.
• Five main areas of hematopathologic molecular testing

  • Detection of mutations, gene rearrangements, and chromosomal abnormalities for diagnosis and prognosis of hematologic malignancies
  • Detection and quantification of minimal residual disease to monitor treatment of hematologic malignancies
  • Detection of mutations in inherited hematologic disorders
  • Pharmacogenetic testing to detect genetic variation affecting certain drug therapies
  • Identification of hematologically important infectious diseases
• Most of the stored information needed to carry out cell processes resides in deoxyribonucleic acid (DNA); therefore proper cellular storage, maintenance, and replication of DNA are necessary to ensure homeostasis.
• Central dogma in genetics

  1. DNA is replicated to daughter DNA

  2. Transcribed to messenger ribonucleic acid (mRNA)

  3. Translated into a functional protein
• In eukaryotes the initial DNA sequence is composed of translated exons separated by untranslated introns. The introns are enzymatically excised following transcription from DNA to RNA, and the mature mRNA sequence is then translated.
• Translation is an enzymatic process wherein mRNA three-nucleotide base sequences, called codons, drive the addition of encoded amino acids to the growing peptide.
• Gene


  The structural units that carry DNA's message
• The human ẞ-globin gene (SBB), part of the hemoglobin molecule, provides a good example of replication and transcription, because it was one of the first sequenced and demonstrates the result of aberrant sequence maintenance.
• In hemoglobin S (Hb S) one inherited mutation changes a single DNA base. This is called a point mutation. The mutation occurs in the sequence that codes for the sixth amino acid of ẞ-globin, and it substitutes the amino acid valine for glutamic acid in the growing peptide.
• Valine modifies the overall charge, producing a protein that polymerizes in a low-oxygen environment. This leads to sickled erythrocytes, circulatory ischemia and its sequelae, and chronic hemolytic anemia.
• Human somatic (nongamete) cells contain 20,000 to 25,000 genes in 2 meters of DNA, with approximately 3 billion DNA base pairs.
• DNA is a duplex molecule composed of two complementary, hydrogen-bonded nucleotide strands.
• Deoxyribonucleotides and ribonucleotides


  The building blocks of DNA and RNA, respectively
• Nucleotide


  Composed of a 5-carbon sugar (pentose), a nitrogenous base, and a phosphate group
• Nucleoside


  A sugar, whether ribose or deoxyribose, linked to a nitrogenous base but without a phosphate group
• To be incorporated into a growing strand of DNA, the nucleotide must have three phosphate groups linked to one another, referred to as α-, ß-, and y-phosphates, with the a-phosphate linked to the sugar.
• Synthesis of a DNA strand

  1. Requires the enzyme DNA polymerase

  2. DNA polymerase recognizes the hydroxyl group on the 3' carbon of the deoxyribose

  3. Forms a phosphodiester bond between the 3' hydroxyl group of the last nucleotide and the 5' a-phosphate group of the next nucleotide to be added
• DNA consists of two strands that are antiparallel and complementary.
• Purines and pyrimidines


  The two categories of nitrogenous bases in nucleic acids
• Adenine (A) and guanine (G) are double-ringed purines, whereas thymine (T) and cytosine (C) are single-ringed pyrimidines.
• Adenine forms hydrogen bonds at two points with thymine (A:T), whereas guanine forms hydrogen bonds at three points with cytosine (G:C).
• In RNA the pyrimidine uracil (U) takes the place of thymine and can form hydrogen bonds with adenine.
• Adenine


  Forms hydrogen bonds at two points with thymine
• Guanine


  Forms hydrogen bonds at three points with cytosine
• 5'-CTAG-3' sequence


  Complementary nucleotides on 3' to 5' strand are 3'-GATC-5'
• Hydrogen bonds between A:T and G:C


  • Hold the DNA strands together
• RNA


  Pyrimidine uracil (U) takes the place of thymine and can form hydrogen bonds with adenine
• DNA


  Resembles a ladder, with the repeating sugar and phosphate groups forming the sides and the bases forming the rungs
• DNA


  Pairing of a double-ringed purine on one strand with a single-ringed pyrimidine on the other maintains a consistent distance between the strands, allowing DNA to twist into a helix
• Transcription


  Conversion of the DNA nucleotide code to mRNA by the enzyme RNA polymerase
• Promoters


  Initiation sequences that lie upstream of coding sequences and bind RNA polymerase
• Translation


  Ribosomes translate the mRNA code into a peptide sequence