Components of a PCR reaction:

1. A DNA template
2. Heat stable Taq DNA polymerase (derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature of 90c, so is not denatured by the high temperatures)
3. Nucleotides
4. A pair of DNA primers
5. Buffers containing magnesium ions

Comparison of PCR components with photocopier items

The principle of PCR is as follows:

1. Starting with a sample of the DNA (template) to be amplified, add the 4 nucleotides (deoxyribonucleoside triphosphates) and the enzyme heat stable DNA polymerase to the solution.

2. Normally (in vivo) the DNA double helix would be separated by the enzyme helicase, but in PCR (in vitro) the strands are separated by heating to 95 c for 2 minutes. This breaks the hydrogen bonds between the 2 DNA strands.

3. Initiation of DNA polymerisation always requires short lengths of DNA (about 20 bp long) called primers. The primers are 2 synthetic oligonucleiotides: one is complementary to a short sequence in one strand of the DNA to be amplified, and the other is complementary to a sequence in the other DNA strand. In vivo the primers are made during replication by DNA polymerase, but in vitro they must be synthesized separately and added at this stage. This means that a short length of the sequence of the DNA must already be known.

4. The DNA must be cooled to 40 c to allow the primers to anneal to their complementary sequences on the separated DNA strands.

5. The DNA polymerase enzyme can now extend the primers and complete the replication of the rest of the DNA. Its optimum temperature is about 72 c, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible.

5. Each original DNA molecule has now been replicated to form 2 molecules. The cycle is repeated from step 2, each time doubling the number of DNA molecules. This is why it is called a chain reaction, since the number of molecules increases exponentially. Typically PCR is run for 20–30 cycles.

PCR can be completely automated; a minute sample of DNA can be amplified millions of times with little effort.

Description of the schematic diagram for PCR technique: Strand 1 and Strand 2 are original DNA strands. The short dark blue fragments are the primers. After multiple heating and cooling cycles, the original strands remain, but most of the DNA consists of amplified copies of the segment (shown in lighter blue) synthesized by the heat stable DNA polymerase.

Uses of PCR:

1. It is appropriate for forensic testing procedures because only a very small sample of DNA is required as the starting material which can be obtained from a single strand of hair or a single drop of blood or semen.

2. Tissue typing for organ transplantation

3. DNA based phylogeny or functional analysis of genes useful for research

4. Detection of genetic diseases: Prenatal genetic diagnosis, Hereditary diseases

5. Detection of infectious diseases: HIV, TB, etc.

6. Parental testing, where an individual is matched with their close relatives

Disadvantage of PCR:

A potential problem for PCR is obtaining a pure sample of DNA to start with; any contaminant DNA will also be amplified.

Use this animation for better understanding of PCR technique from DNA Learning Center.

Sources:

• Essential Biochemistry for Medicine
• Mark’s Medical Biochemistry

Further Reading:
Isoenzymes and their Diagnostic Importance

• Also called isozymes
• Are multiple forms of an enzyme that catalyzes the same reaction
• Arise through gene duplication
• Differ in their physical and chemical properties, Km and Vmax values, optimum pH, substrate affinity, etc.

Lactate Dehydrogenase (LDH)

Lactate ←LDH→ Pyruvate

LDH is a tetrameric enzyme with 2 types of subunit “H” and “M” :

1. M (for muscle) : basic
2. H (for heart) : acidic

Isoenzymes of LDH:

1. LDH1 (H4) : Heart and RBC
2. LDH2 (H3M) : Heart and RBC
3. LDH3 (H2M2) : Brain and kidney
4. LDH4 (HM3) : Liver and skeletal muscle
5. LDH5 (M4) : Liver and skeletal muscle

LDH1 has high Km (low affinity) and LDH5 has low Km (high affinity) for pyruvate.

Diagnositc importance of LDH:

1. Normal: LDH2 > LDH1
2. Myocardial infarction (within 12-24 hours): LDH1>>LDH2 (flipped LDH pattern)
3. Liver diseases: increased LDH5 in serum
4. Increased LDH suggests other following diseases:
   • Hemolytic anemia
   • Hypotension
   • Infectious mononucleosis
   • Intestinal ischemia and infarction
   • Muscle injury
   • Muscular dystrophy
   • Pancreatitis
   • Lung tissue death
   • Stroke
   • Ischemic cardiomyopathy

Creatine Phosphokinase (CPK)

Phosphocreatine ←CPK→ Creatine

CPK is a dimeric enzyme consisting of 2 subunits:
a. M : for muscle
b. B : for brain

Isoenzymes of CPK:

1. CPK1 (BB) : Brain
2. CPK2 (MB) : Heart
3. CPK3 (MM) : Skeletal muscles

Diagnositic importance of CPK:

1. Normal: low CPK2 (<2%) in serum
2. MI (within 6-18 hrs): increased CPK2 (20%) in serum
3. Increased CPK-1 suggests:
   • Brain cancer
   • Brain injury
   • Pulmonary infarction
   • Seizure
4. Increased CPK-2 suggests other diseases like:
   • Electrical injuries
   • Heart injury
   • Myocarditis
5. Increased CPK-3 suggests:
   • Crush injuries
   • Rhabdomyolysis
   • Muscular dystrophy
   • Myositis
   • Recent seizures

Alkaline Phosphatase (ALP):

The enzyme is a monomer and the isoenzymes are due to the difference in the carbohydrate content:

1. Alpha1-ALP
2. Alpha2-Heat labile ALP
3. Alpha2-Heat stable ALP
4. Pre Beta-ALP
5. Gamma-ALP ,etc.

Diagnostic importance of ALP:

1. Increased Alpha2-Healt labile ALP:
   • Liver diseases : Biliary obstruction, hepatitis
2. Increased pre beta-ALP:
   • Bone diseases: Paget’s disease, Osteoblastic bone tumors, Osteomalacia, Rickets, Skeletal disease
3. Increase in ALP also suggests other diseases like:
   • Pregnancy
   • Healing bone fracture
   • Anemia
   • Leukemia
   • Thyroid gland inflammation
   • Hyperparathyroidism
   • Chronic alcohol ingestion
4. Decreased ALP suggests:
   • Malnutrition

Sources:

• Harper’s Illustrated Biochemistry
• Biochemistry – Satyanarayana
• Medline Plus : Isoenzyme tests

Definition:

CPT I (Carnitine palmitoyltransferase I) deficinecy is a rare autosomal recessive metabolic disorder that prevents the body from converting certain fats called long chain fatty acids into energy and is generally associated with a viral illness and prolonged fasting. Onset is in infancy or early childhood.

Cause:

Homozygous mutation in the CPT1A (CPT1) gene that provides instructions for making a liver enzyme called carnitine palmitoyl transferase I. Carnitine is required by cells to process fats and produce energy but people with this disorder have a faulty enzyme that disrupts carnitine’s role in processing long chain fatty acids.

CPT1A: Function and Disorder

Effects (Symptoms):

• Hypoketotic hypoglycemia
• Encephalopathy
• Hepatomegaly
• Elevated blood level of carnitine
• Muscle weakness
• Nervous system damage
• Mild metabolic acidosis with or without lactic acidemia
• Hyperammonemia
• Loss of consciousness
• Seizures
• Low blood sugar
• Elevated trnasaminases
• Lethargy
• Urinary ketones are absent
• Behavior changes and irritable mood
• Poor appetite
• Fever, diarrhea, vomiting
• Untreated babies have learning problems

Diagnosis:

• Newborn screening of the heel stick dried blood spot using tandem mass spectrometry finds elevation of free carnitine and reduction of long-chain acylcarnitines (i.e. C16:0 and C18:0).
• The definitive diagnosis of CPT I deficiency is made by measuring enzyme activity in fibroblasts, leukocytes or liver. A variety of mutations have been detected in the gene for hepatic CPT I, but no common mutations have been found to allow easy DNA diagnosis.

This condition is sometimes mistaken for Reye’s Syndrome.

Treatment:

• Avoid prolonged fasting
• Carbohydrate and protein rich diet (low fat)
• Medium chain triglyceride (MCT) oil supplement
• Administration of IV glucose during acute episodes