PCR, A Surge Forward

What is it?

PCR or polymerase chain reaction is a technique used in molecular biology and diagnostics to amplify a single or few copies of a sequence of DNA or RNA into thousands to millions. Developed in 1983 by Kary Mullis, who also went on to win the Nobel Prize in 1993.

It changed the way for a lot of different domains. PCR is an indispensable technique used in clinical and research laboratories with a broad span of applications. PCR is used for DNA cloning, gene cloning and manipulation, gene mutagenesis and functional analysis of genes for diagnostic or monitoring purposes.

PCR is dependent on thermal cycling; that is exposing the reactants to cycles of repeated heating and cooling which allows different temperature dependent reactions to take hold.

So, how does it work?

The basic PCR set-up requires several specific components and reagents. There needs to be a DNA template that contains the DNA target sequence that is targeted for amplification. DNA polymerase is needed, more specifically taq polymerase as it is heat-resistant. Taq polymerase is an enzyme that is isolated from the thermophilic bacterium Thermus aquaticus. It can survive the high-temperature DNA denaturation phase. Primers that are complementary to the 3′ ends of each sense and anti-sense strands of the DNA target. Primers are specific and complementary to the target sequence and are often selected beforehand. More than likely the primers are artificially synthesized from a commercial biochemical supplier. Deoxynucleoside triphosphates of dNTPs are the building blocks from which taq polymerase synthesizes a new strand.

There are three steps to PCR; The first step, denaturation is the first step in the natural cycle of events and consists of heating the reaction to 94-98 degrees Celsius for 20-30 seconds. This causes DNA denaturation of the dsDNA template by breaking the hydrogen bonds between the complementary base-pairs. The result is two, ssDNA molecules. Annealing is the second step in PCR and the temperature is lowered to 50-65 degrees Celsius (122-149 F) for 20-40 seconds. This allows annealing of the primers to each of the ssDNA molecules. The temperature in the annealing step is critical because you must select a temperature low enough to allow hybridization of the primer to the strands, but high enough for the hybridization to be specific to the target sequence selected to amplify. The primer should only bind to the complementary part of the strand and no where else. If the temperature is too low the primer may bind improperly and cause issues or may not bind at all. Extension and elongation is the final step in PCR where the DNA taq polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture in a 5′ to 3′ direction. The reaction is raised to about 72 degrees Celsius. The 5′-phosphate group is condensed to the 3′-hydroxyl group at the end of the nascent or elongating new DNA strand.

Polymerase_chain_reaction

At the elongation step in each cycle, the number of DNA copies is doubled. Denaturation, annealing, and elongation constitute a single cycle.

PCR can fail for various reasons. PCR is very sensitive to contamination causing DNA amplification of erroneous DNA products. Primer-design techniques are important to improving PCR product yield and in avoiding the production of wrong DNA products. There are multiple primer rules that should be followed; Primers should be between 22-26 bases in length with optimization at 24. Specificity and Tm (Melting temperature) should be between 58-66 degrees Celsius. Both primers should have a similar Tm (+/- 2 degrees). Keep the G-C base pair content between 40-60% , optimization at 50% if possible. Avoid repetitive sequences (AAAA, TATATA) as they can cause mis-priming. Because annealing of the primer is most critical at its 3′ end, a primer that has a high G-C content at its 3′ end is more likely to cause mis-priming.

Some notable analogs of PCR are RT-PCR (Reverse transcriptase PCR) and qPCR (quantitative PCR). RT-PCR is used for amplifying DNA from RNA. Reverse transcriptase enzyme transcribes RNA template into cDNA, which is then amplified. This is widely used for gene expression profiling or to identify the sequence of an RNA transcript. This determines the expression profile of a gene if known. It can be used to map the exons and introns in the gene. qPCR is used to measure the quantity of the target sequence. It quantitatively measures starting amounts of DNA, cDNA, or RNA and measures the amount of copies in the sample. qPCR is highly specific and precise. Usually qPCR methods use fluorescent dyes, most commonly Sybr green or Taqman which measures the amount of amplified product in real time.

PCR and its PCR derivatives have evolved and changed the way clinical labs and diagnostics are performed. There is a wide range of use in medicine.

Check out my previous posts;

Gram-negative diplococci failed to grow at 48 hours in 35 degree Celsius.

FAB Classification of Leukemias and Cytochemical Stain Observations in each.

3 year old evaluated with abdominal pain and anorexia.

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Gram-negative diplococci failed to grow at 48 hours in 35 degree Celsius.

An urethral swab was obtained from a man with an urethral exudate was plated directly on chocolate agar and modified Thayer-Martin agar and a gram stain was made. The culture plates were incubated at 35 degrees celsius, but showed no growth at 48 hours. The gram stain showed gram-negative diplococci.

What is a Thayer-Martin agar? A Thayer-Martin agar is a Mueller-Hinton agar with 5% chocolate sheep blood and a combination of antibiotics. The antibiotics include vancomycin, colistin, nystatin, and trimethoprim. Vancomycin kills most gram-positive organisms, except Lactobacillus and Pediococcus species are inherently resistant. Colistin is added to kill most gram-negative organisms except Neisseria. Nystatin is added to kill most fungi. Trimethoprim inhibits gram-negative organisms especially Proteus species.

A Thayer-Martin agar is used to isolate suspected pathogenic Neisseria gonorrhoeae and meningitidis.

A chocolate blood agar (CBA, CHOC) is a nonselective enriched growth media used for isolation of pathogenic bacteria. The chocolate agar is used for growing fastidious respiratory bacteria such as Haemophilus influenzae or Neisseria meningitidis. Some of the fastidious bacteria need NAD and hemin which are inside RBCs, which are lysed in the chocolate agar.

Typically when a pathogenic Haemophilus or Neisseria is suspected, the swab will first be plated onto a chocolate agar, and then a more selective media, either being the chocolate agar with the addition of bacitracin for selection of Haemophilus, or the Thayer-Martin agar for the selection of Neisseria species.

Because of the source of the swab, being from a urethral exudate, this makes Neisseria more likely so sample was restreaked onto a Thayer-Martin agar.

Neisseria gonorrhoeae is a gram-negative diplococci (which was observed on upon gram stain). It is oxidase positive, aerobic, and non-mobile. It survives within PMNs within the host and is the causative agent of gonorrhea.

The reason that it failed to grow at 35 degrees celsius after 48 hours is because Neisseria gonorrhoeae requires an environment enriched with 3-7% carbon dioxide.  This species is capnophilic meaning that it strives in higher levels of carbon dioxide.

A way to differentiate Neisseria gonorrhoeae from Neisseria meningitidis is from a carbohydrate utilization test. N. gonorrhoeae ONLY oxidizes glucose while N. meningitidis is able to ferment glucose and maltose.

 

BOC Online Study Guide; Microbiology

 

FAB Classification of Leukemias and Cytochemical Stain Observations in each.

Cytochemical stains are great tools to distinguish the different myelogenous leukemias from each other. There are multiple different stains employed to help this process.

Myeloperoxidase (MPO): MPO is a lysosomal protein that is stored in the azurophilic granules of neutrophils and is released upon degranulation into the extracellular space. During respiratory burst it produces hypochlorous acid from hydrogen peroxide and chloride.

Sudan Black B: SBB is a lysochrome (fat-soluble dye) that is used for staining the lipids and lipoproteins. This is helpful in differentiating between AML and ALL as SBB will only stain myeloblasts and not lymphoblasts.

A-Napthyl-Chloroacetate: Otherwise known as the specific esterase stain will stain red in granulocytes and immature granulocytes.

A-Napthyl-acetate/butyrate: Otherwise known as the non-specific esterase stain will reveal strong staining patterns in monocytic lineage cells and will not stain lymphoblasts or myeloblasts.

PAS: Periodic acid–Schiff (PAS) is a staining method used to detect polysaccharides such as glycogen, and mucosubstances such as glycoproteins, glycolipids and mucins in tissues. Erythroleukemia, a leukemia of immature red blood cells will stain a bright fuchsia.

LAP: Leukocyte alkaline phosphatase (LAP) is the term for alkaline phosphatase that’s found in leukocytes. This can differentiate other WBC disorders from CML. In CML the LAP is decreased.

M0: Acute myeloid leukemia minimally differentiation; No stains positive, It can be possible for a weak MPO or SBB reaction.

M1: Acute myeloid leukemia without maturation; Weakly MPO and SBB positive.

M2: Acute myeloid leukemia with maturation; MPO and SBB positive

M3: Acute promyelocytic leukemia; MPO and SBB strongly positive

M4: Acute myelomonocytic; MPO and SBB positive. Non-specific and specific esterase stain positive.

M5: Acute monocytic/monoblastic leukemia; Non-specific esterase stain positive.

M6: Acute erythroleukemia; PAS positive

M7: Acute megakaryoblastic leukemia. Antibody reactions are positive. Usually anti-VIII, and Anti-GP IIb/IIIa.

None of these stains will yield a definitive diagnosis and are to be used in conjunction with other tests to make a diagnosis of AML.

3 year old evaluated with abdominal pain and anorexia.

A three year old child was evaluated for abdominal pain and anorexia by a physician. CBC reveals Hgb of 9.8 g/dL and basophilic stippling of the RBCs.

The physician should order further tests to check for poisoning from?

a) Arsenic

b) Iron

c) Mercury

d) Lead

Patient presents slightly anemic with a hemoglobin of 9.8 g/dL (Reference children: 11.0-14.5 g/dL). Basophilic stippling is residual ribosomal RNA that can be seen on a wrights stain. It can be observed in some Myelodysplastic disorders, sideoblastic anemia, megaloblastic anemia, arsenic poisoning, and thalassemia.

The correct answer is lead poisoning. Lead interferes with heme synthesis. It inhibits ALA dehydratase otherwise known as porphobilinogen synthase (PBG) which stops heme synthesis. Other findings consistent with lead poisoning would be a microcytic, and hypochromic RBC.

Elevated lead levels in a whole blood lead test is diagnostic. Urine D-ALA and RBC zinc protoporphyrin are also useful assays.

Children are at more of risk for lead poisoning because they are in a continuous state of growth and development and lead is absorbed at a faster rate than in adults. The classical signs in children are loss of appetite, abdominal pain, vomiting, weight loss, constipation, anxiety, kidney failure, irritability, growth retardation, and behavior problems. Sometimes permanent intellectual disability can be seen. As you can see lead poisoning can affect multiple organ systems.

Lead can come from from anywhere, the most common exposure route in adults is occupational exposure. Lead can also be found in food if the food is grown in soil that is high in lead. It can be found in paint, in the soil, and in water sources.

 

BOC Study Guide 5th Edition, ASCP, pgs. 115,138

What Is Medical Laboratory Science?

According to the American Society for Clinical Laboratory Science (ASCLS) medical scientists are vital in healthcare, uncovering and providing important laboratory analyses that assist physicians in patient diagnosis and treatment. Scientists have a role in monitoring disease progression and treatment monitoring as well.

Laboratory scientists utilize technologically advanced biomedical instrumentation and computers to perform laboratory testing across a wide range of disciplines. Such disciplines include clinical chemistry, hematology, immunology, immunohematology, microbiology and molecular biology.

Without clinical laboratory scientists Physicians would not be able to see patients, they would not be able to test them or treat them. A hospital really runs on the expertise of its laboratory professionals. The behind the scenes work done by the lab professionals is just as important as the patient interaction done by other healthcare professionals.