Updated: 1/25/2018

Polymerase Chain Reaction (PCR)

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PCR overview
  • Function 
    • can amplify a selected region of DNA 
  • Process 
    • solution prepared containing
      • DNA primers specific for selected DNA region
      • DNA sample of interest
      • heat stable DNA polymerase
      • deoxyribonucleotides
    • denaturation of dsDNA by heating
    • annealing of DNA primer specific for region of interest and slowly cooling the solution
    • replication of DNA at the primer by heat stable DNA polymerase
    • repetition of the process several times 
    • gel electrophoresis used to separate the various components of the solution
  • Clinical use
    • in all uses PCR functions to amplify the amount of DNA present in a sample
    • high specificity bacterial/viral infection testing 
      • HIV
        • first step is ELISA (high sensitivity)
        • PCR is used to determine viral load
        • test examines the amount of viral DNA integrated into host cell DNA
        • advantages over ELISA
          • PCR becomes positive earlier in disease course
            • Positive ELISA result is dependent on antibody formation
          • PCR does not require that the patient have a competent immune system
            • ELISA requires the host to make antibodies
        • important specific cases when PCR should always be used
          • a newborn whose mother is HIV+
            • will have antibodies even if not infected, so ELISA does not work
          • when earliest possible detection is required
    • genetic identification
      • forensic/paternity testing
      • use of variable number tandem repeats (VNTRs) or short tandem repeats (STRs)
        • unique copies of non-coding regions of DNA between individuals
        • since they exist on both chromosomes, individuals have two copies at each locus
          • 1 paternal and 1 maternal
      • can only prove with certainty that the sample DOES NOT belong to the test subject
        • cannot prove with 100% certainty that DNA belongs to individual of interest because there is a small chance that someone shares the same VNTR or STR
    • direct mutation
      • if DNA region is known, PCR can amplify that region for sequencing
RT-PCR overview
  • Function
    • used to measure the amount of RNA present in a sample
  • Process
    • reverse transcriptase is added to solution containing RNA, dNTPs, primers for specific sequence of interest, and heat stable DNA polymerase
    • RNA is converted to DNA and DNA sequence of interest is amplified
    • When combined with quantitative (real-time PCR), the combined qRT-PCR (quantitative reverse transcription PCR) assesses the amount of RNA in the original sample by measuring the amount of amplified PCR product after a set number of PCR cycles, which is directly proportional to the concentration of RNA in the original sample
  • Clinical use
    • HIV viral load
      • measures transcriptional activity of the virus by detecting the amount of RNA present
      • gives a more detailed picture of the infection and treatment results
 

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Questions (4)
Lab Values
Blood, Plasma, Serum Reference Range
ALT 8-20 U/L
Amylase, serum 25-125 U/L
AST 8-20 U/L
Bilirubin, serum (adult) Total // Direct 0.1-1.0 mg/dL // 0.0-0.3 mg/dL
Calcium, serum (Ca2+) 8.4-10.2 mg/dL
Cholesterol, serum Rec: < 200 mg/dL
Cortisol, serum 0800 h: 5-23 μg/dL //1600 h:
3-15 μg/dL
2000 h: ≤ 50% of 0800 h
Creatine kinase, serum Male: 25-90 U/L
Female: 10-70 U/L
Creatinine, serum 0.6-1.2 mg/dL
Electrolytes, serum  
Sodium (Na+) 136-145 mEq/L
Chloride (Cl-) 95-105 mEq/L
Potassium (K+) 3.5-5.0 mEq/L
Bicarbonate (HCO3-) 22-28 mEq/L
Magnesium (Mg2+) 1.5-2.0 mEq/L
Estriol, total, serum (in pregnancy)  
24-28 wks // 32-36 wks 30-170 ng/mL // 60-280 ng/mL
28-32 wk // 36-40 wks 40-220 ng/mL // 80-350 ng/mL
Ferritin, serum Male: 15-200 ng/mL
Female: 12-150 ng/mL
Follicle-stimulating hormone, serum/plasma Male: 4-25 mIU/mL
Female: premenopause: 4-30 mIU/mL
midcycle peak: 10-90 mIU/mL
postmenopause: 40-250
pH 7.35-7.45
PCO2 33-45 mmHg
PO2 75-105 mmHg
Glucose, serum Fasting: 70-110 mg/dL
2-h postprandial:<120 mg/dL
Growth hormone - arginine stimulation Fasting: <5 ng/mL
Provocative stimuli: > 7ng/mL
Immunoglobulins, serum  
IgA 76-390 mg/dL
IgE 0-380 IU/mL
IgG 650-1500 mg/dL
IgM 40-345 mg/dL
Iron 50-170 μg/dL
Lactate dehydrogenase, serum 45-90 U/L
Luteinizing hormone, serum/plasma Male: 6-23 mIU/mL
Female: follicular phase: 5-30 mIU/mL
midcycle: 75-150 mIU/mL
postmenopause 30-200 mIU/mL
Osmolality, serum 275-295 mOsmol/kd H2O
Parathyroid hormone, serume, N-terminal 230-630 pg/mL
Phosphatase (alkaline), serum (p-NPP at 30° C) 20-70 U/L
Phosphorus (inorganic), serum 3.0-4.5 mg/dL
Prolactin, serum (hPRL) < 20 ng/mL
Proteins, serum  
Total (recumbent) 6.0-7.8 g/dL
Albumin 3.5-5.5 g/dL
Globulin 2.3-3.5 g/dL
Thyroid-stimulating hormone, serum or plasma .5-5.0 μU/mL
Thyroidal iodine (123I) uptake 8%-30% of administered dose/24h
Thyroxine (T4), serum 5-12 μg/dL
Triglycerides, serum 35-160 mg/dL
Triiodothyronine (T3), serum (RIA) 115-190 ng/dL
Triiodothyronine (T3) resin uptake 25%-35%
Urea nitrogen, serum 7-18 mg/dL
Uric acid, serum 3.0-8.2 mg/dL
Hematologic Reference Range
Bleeding time 2-7 minutes
Erythrocyte count Male: 4.3-5.9 million/mm3
Female: 3.5-5.5 million mm3
Erythrocyte sedimentation rate (Westergren) Male: 0-15 mm/h
Female: 0-20 mm/h
Hematocrit Male: 41%-53%
Female: 36%-46%
Hemoglobin A1c ≤ 6 %
Hemoglobin, blood Male: 13.5-17.5 g/dL
Female: 12.0-16.0 g/dL
Hemoglobin, plasma 1-4 mg/dL
Leukocyte count and differential  
Leukocyte count 4,500-11,000/mm3
Segmented neutrophils 54%-62%
Bands 3%-5%
Eosinophils 1%-3%
Basophils 0%-0.75%
Lymphocytes 25%-33%
Monocytes 3%-7%
Mean corpuscular hemoglobin 25.4-34.6 pg/cell
Mean corpuscular hemoglobin concentration 31%-36% Hb/cell
Mean corpuscular volume 80-100 μm3
Partial thromboplastin time (activated) 25-40 seconds
Platelet count 150,000-400,000/mm3
Prothrombin time 11-15 seconds
Reticulocyte count 0.5%-1.5% of red cells
Thrombin time < 2 seconds deviation from control
Volume  
Plasma Male: 25-43 mL/kg
Female: 28-45 mL/kg
Red cell Male: 20-36 mL/kg
Female: 19-31 mL/kg
Cerebrospinal Fluid Reference Range
Cell count 0-5/mm3
Chloride 118-132 mEq/L
Gamma globulin 3%-12% total proteins
Glucose 40-70 mg/dL
Pressure 70-180 mm H2O
Proteins, total < 40 mg/dL
Sweat Reference Range
Chloride 0-35 mmol/L
Urine  
Calcium 100-300 mg/24 h
Chloride Varies with intake
Creatinine clearance Male: 97-137 mL/min
Female: 88-128 mL/min
Estriol, total (in pregnancy)  
30 wks 6-18 mg/24 h
35 wks 9-28 mg/24 h
40 wks 13-42 mg/24 h
17-Hydroxycorticosteroids Male: 3.0-10.0 mg/24 h
Female: 2.0-8.0 mg/24 h
17-Ketosteroids, total Male: 8-20 mg/24 h
Female: 6-15 mg/24 h
Osmolality 50-1400 mOsmol/kg H2O
Oxalate 8-40 μg/mL
Potassium Varies with diet
Proteins, total < 150 mg/24 h
Sodium Varies with diet
Uric acid Varies with diet
Body Mass Index (BMI) Adult: 19-25 kg/m2
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(M1.BC.1) A 27-year-old HIV positive female gave birth to a 7lb 2oz (3.2 kg) baby girl. The obstetrician is worried that the child may have been infected due to the mother's haphazard use of her anti-retroviral medications. Which of the following assays would a hospital use detect the genetic material of HIV if the child had been infected? Review Topic

QID: 106269
1

Enzyme-linked immunosorbent assay (ELISA)

33%

(7/21)

2

Western blot

5%

(1/21)

3

Rapid HIV antibody test

5%

(1/21)

4

Polymerase chain reaction

57%

(12/21)

5

Southern blot

0%

(0/21)

M1

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(M1.BC.74) Polymerase chain reaction (PCR) has revolutionized the molecular biology field. PCR allows for amplification of a single piece of genetic material to produce trillions of idential copies. Which of the following is NOT necessary for PCR? Review Topic

QID: 106615
1

Taq polymerase

10%

(18/189)

2

Reverse transcriptase

60%

(113/189)

3

Primers

9%

(17/189)

4

Deoxynucleotide triphosphates

15%

(29/189)

5

Target DNA strand

4%

(8/189)

M1

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