Polymerase Chain Reaction ( PCR technology)

Kary Mullis discovered polymerase chain reaction in 1983. PCR is a method employed to amplify minute amount of DNA within a few hours.

This technique consists of selective amplification of specific target nucleic acid sequences from total DNA by using primers specific for the target region to be amplified.

Polymerase Chain Reaction And Its Applications Basic Steps

• Denaturation: In this step, the double-stranded template DNA is denatured by heat (temperature of around 92–96°C) into single-stranded DNA.
• Annealing: DNA primers of interest are added along with the four basic deoxynucleotides and the solution is cooled. It causes binding of DNA probes to their specific target regions of the single-stranded DNA at a temperature of around 50–65°C.
• Extension: The primers are extended at a temperature of around 68–78°C in the presence of DNA polymerase, dNTPs and Mg2+ ions.

The newly synthesized DNA strand acts as a template for the next cycle.

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This cycle is repeated several times (around 25–30 times) and produces millions of copies of the original specific target DNA.

To retain the activity of DNA polymerase enzyme at such high denaturation temperature, Taq DNA polymerase, extracted from a microorganism (Thermus aquaticus) is used in the PCR reaction.

Applications of Polymerase Chain Reaction

Polymerase chain reaction is the starting test used in most of the molecular genetic tests.

• Diagnosis of hereditary/genetic diseases: In genetic disorders, genetic mutations can be detected by PCR alone (e.g., Huntington disease), PCR followed by digestion with restriction endonucleases (e.g., diagnosis of spinal muscular atrophy), PCR followed by dot-blot hybridization (e.g., thalassemia mutation detection), PCR followed by capillary electrophoresis for genotyping (e.g., detection of triplet repeat disorders).
• Sequencing: It is used as first step for DNA amplification for all methods of DNA sequencing.
• Detection of pathogens: To detect small quantities of pathogen DNA (e.g., PCR for Mycobacterium tuberculosis), to detect viruses, (including use of reverse transcriptase enzyme for RNA viruses), and real-time PCR (RT-PCR) for quantification
  of viral load, e.g., hepatitis B and C, human immunodeficiency virus (HIV).
• Forensic genetics: To identify DNA sequences that are unique to each individual.

Reverse transcriptase PCR (RT-PCR): Initial step is to convert RNA to complementary DNA and rest of the process is same as PCR. It is used to quantify the amount/number of copies of input DNA/RNA.

Real-time PCR: This technique, quantifies the PCR product in “real-time”. It is used to quantify the amount/number of copies of input DNA/RNA.

Immunoblot (Western Blot)

It is a test to detect antibodies. According to molecular weight, the microbial proteins are separated by polyacrylamide gel electrophoresis (PAGE).

They are transferred (blotted) on to a nitrocellulose membrane, which is incubated with serum of patient.

Binding of specific antibody is detected with an enzyme—anti-immunoglobulin conjugate (similar to in ELISA), and specificity is confirmed by its location on the membrane.

The test is a highly specific and can be used to confirm the results of less specific tests such as ELISA.

Lysosomal Storage Disorders

Classifiation of Lysosomal Storage Disorders

The classification of lysosomal storage disorders has been

Lysosomal Storage Disorders Mucopolysaccharidoses

The various types of mucopolysaccharidoses

MPS I (Hurler Syndrome)

• Head/face: Prominence of the forehead, broad nose with a flattened nasal bridge, enlargement and protrusion of the tongue, chronic hearing loss, corneal clouding, retinal degeneration or glaucoma; tonsillar/adenoidal hypertrophy (predispose the child to URI), sleep apnea.
• Heart: Cardiomyopathy with asymmetric hypertrophy of the ventricular septum, thickening of the aortic and mitral valves, which progress to valvular insufficiency.
• Abdomen: Hepatosplenomegaly results in a protuberant abdomen.
• Neurological: Mental retardation.

MPS II (Hunter Syndrome)

• Initial symptoms starts at 2–4 years of age with progressive growth delays, resulting in short stature.
• Head/face: Macrocephaly, delayed tooth eruption, progressive hearing loss, thickening of the lips, tongue, and nostrils.
• Heart: Thickening of the heart valves leading to a decline in cardiac function.
• Lungs: Obstructive airway disease.
• Abdomen: Hepatosplenomegaly.
• Skeletal: Short neck and broad chest, joint stiffness, with restriction of movements.
• Neurological: Hydrocephalus, mental retardation, and seizures.

MPS IV (Morquio Syndrome)

• Widely spaced teeth, corneal clouding hearing loss, enlarged heart.
• Abnormal skeletal development: Scoliosis, hypermobile joints, large fingers, knock-knees, short trunk with pectus carinatum, bell-shaped chest (ribs flared), severe growth retardation (82–115 cm), odontoid hypoplasia, compression of spinal cord (cervical myelopathy), and dwarfism.

Lysosomal Storage Disorders Phenylketonuria (PKU)

Autosomal recessive disease, due to deficiency of enzyme phenylalanine hydroxylase causing failure to convert phenylalanine to tyrosine.

Developmental delay, mental retardation, seizures, eczema, blue eyes, hyperactivity, aggressive behavior, blond hair and musty/mousy odor.

Lysosomal Storage Disorders Galactosemia

Autosomal recessive disease due to deficiency of the enzyme galactose-1-phosphate uridyltransferase. It is due to ingestion of galactose (lactose).

• Liver failure (hy poglycemia, bilirubinemia), hepatosplenomegaly
• Renal tubular disorder (acidosis, glycosuria, albuminuria)
• Lethargy, feeding intolerance, failure to thrive, cataracts
• Learning disorders in older children—mental retardation
• About 25% will develop sepsis (E. coli) in first 1–2 weeks, if untreated → death.