Polymerase chain reaction (PCR )

Polymerase Chain Reaction (PCR)- Principle, Steps, Applications

PCR is an enzymatic process in which a specific region of DNA is replicated over and over again to yield many copies of a particular sequence.
The most widely used target nucleic acid amplification method is the polymerase chain reaction (PCR).
This method combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication applied repeatedly through numerous cycles.
This method is able to amplify a single copy of a nucleic acid target, often undetectable by standard hybridization methods, and multiply to 10⁷or more copies in a relatively short period.
This thus provides ample target that can be readily detected by numerous methods.

Principle of PCR

The target sequence of nucleic acid is denatured to single strands, primers specific for each target strand sequence are added, and DNA polymerase catalyzes the addition of deoxynucleotides to extend and produce new strands complementary to each of the target sequence strands (cycle 1). In cycle 2, both double-stranded products of cycle 1 are denatured and subsequently serve as targets for more primer annealing and extension by DNA polymerase. After 25 to 30 cycles, at least 10⁷ copies of target DNA may be produced by means of this thermal cycling.

Requirements for PCR

A PCR reaction contains the target double-stranded DNA, two primers that hybridize to flanking sequences on opposing strands of the target, all four deoxyribonucleoside triphosphates and a DNA polymerase along with buffer, co-factors of enzyme and water.
Since the reaction periodically becomes heated to high temperature, PCR depends upon using a heat-stable DNA polymerase.
Many such heat-stable enzymes from thermophilic bacteria (bacteria that live in high temperature surroundings) are now available commercially.
The first one and the most commonly used is the Taq polymerase from the thermophilic bacterium Thermus aquaticus.

Steps Involved

Polymerase Chain Reaction

A. Extraction and Denaturation of Target Nucleic Acid

For PCR, nucleic acid is first extracted (released) from the organism or a clinical sample potentially containing the target organism by heat, chemical, or enzymatic methods.
Once extracted, target nucleic acid is added to the reaction mix containing all the necessary components for PCR (primers, nucleotides, covalent ions, buffer, and enzyme) and placed into a thermal cycler to undergo amplification.

B. Steps in Amplification

Conventional PCR involves 25 to 50 repetitive cycles, with each cycle comprising three sequential reactions:

Denaturation of target nucleic acid
Primer annealing to single-strand target nucleic acid extension of primer target duplex.
Extension of the primer-target duplex.

Denaturation

The reaction mixture is heated to 95°C for a short time period (about 15–30 sec) to denature the target DNA into single strands that can act as templates for DNA synthesis.

Primer annealing

The mixture is rapidly cooled to a defined temperature which allows the two primers to bind to the sequences on each of the two strands flanking the target DNA.
Primers are short, single-stranded sequences of nucleic acid (i.e., oligonucleotides usually 20 to 30 nucleotides long) selected to specifically hybridize (anneal) to a particular nucleic acid target, essentially functioning like probes.
This annealing temperature is calculated carefully to ensure that the primers bind only to the desired DNA sequences (usually around 55^oC).
One primer binds to each strand. The two parental strands do not re-anneal with each other because the primers are in large excess over parental DNA.

Extension

The temperature of the mixture is raised to 72°C (usually) and kept at this temperature for a pre-set period of time to allow DNA polymerase to elongate each primer by copying the single-stranded templates.
Annealing of primers to target sequences provides the necessary template format that allows the DNA polymerase to add nucleotides to the 3’ terminus (end) of each primer and extend sequence complementary to the target template
Taq polymerase is the enzyme commonly used for primer extension, which occurs at 72°C. This enzyme is used because of its ability to function efficiently at elevated temperatures and to withstand the denaturing temperature of 94°C through several cycles.
The ability to allow primer annealing and extension to occur at elevated temperatures without detriment to the polymerase increases the stringency of the reaction, thus decreasing the chance for amplification of non-target nucleic acid (i.e., nonspecific amplification).

The three steps of the PCR cycle are repeated.

Thus in the second cycle, the four strands denature, bind primers and are extended. No other reactants need to be added. The three steps are repeated for a third cycle and so on for a set of additional cycles.
By the third cycle, some of the PCR products represent DNA sequence only between the two primer sites and the sequence does not extend beyond these sites.
As more and more reaction cycles are carried out, the double-stranded DNA are synthesized more in number. After 20 cycles, the original DNA has been amplified a million-fold and this rises to a billion fold (1000) million after 30 cycles.

C. Product Analysis

Gel electrophoresis of the amplified product is commonly employed after amplification.
The amplified DNA is electrophoretically migrated according to their molecular size by performing agarose gel electrophoresis.
The amplified DNA forms clear bands which can be visualized under ultra-raviolet (UV) light.

PCR

Advantages of PCR

PCR (polymerase chain reaction) is an extremely simple yet immensely powerful technique.
It allows enormous amplification of any specific sequence of DNA provided that short sequences either side of it are known.
Allow faster diagnosis and identification while enhancing sensitivity and maintaining specificity.

Applications of PCR

PCR already has very widespread applications, and new uses are being devised on a regular basis.

PCR can amplify a single DNA molecule from a complex mixture, largely avoiding the need to use DNA cloning to prepare that molecule. Variants of the technique can similarly amplify a specific single RNA molecule from a complex mixture.
DNA sequencing has been greatly simplified using PCR, and this application is now common.
By using suitable primers, it is possible to use PCR to create point mutations, deletions and insertions of target DNA which greatly facilitates the analysis of gene expression and function.
PCR is exquisitely sensitive and can amplify vanishingly small amounts of DNA. Thus, using appropriate primers, very small amounts of specified bacteria and viruses can be detected in tissues, making PCR invaluable for medical diagnosis.
PCR is now invaluable for characterizing medically important DNA samples. For example, in screening for human genetic diseases, it is rapidly replacing the use of RFLPs.
Because of its extreme sensitivity, PCR is now fundamentally important to forensic medicine. It is even possible to use PCR to amplify the DNA from a single human hair or a microscopic drop of blood left at the scene of a crime to allow detailed characterization.

Complete Guide on cholesterol

What is cholesterol?

You probably have heard about cholesterol, but you might not be sure exactly what it is. Cholesterol is a waxy type of fat, or lipid, which moves throughout your body in your blood.

If you have too much cholesterol in your blood, it can combine with other substances in the blood to form plaque. Plaque sticks to the walls of your arteries. This buildup of plaque is known as atherosclerosis. It can lead to coronary artery disease, where your coronary arteries become narrow or even blocked.

Why is cholesterol important to our bodies?

Cholesterol is a chemical compound that the body requires as a building block for cell membranes and for hormones like estrogen and testosterone.

Cholesterol is also needed to produce vitamin D. Your liver makes enough cholesterol to meet your body’s needs for these important functions.

The liver produces about 80% of the body's cholesterol and the rest comes from dietary sources like meat, poultry, eggs, fish, and dairy products. Foods derived from plants contain no cholesterol.

What are the types of cholesterol?

Cholesterol doesn’t dissolve in water, so it can’t travel through your blood on its own. To help transport cholesterol, your liver produces lipoproteins.

These lipoprotein include:

· HDL

· LDL

· VLDL

HDL stands for high-density lipoprotein. It is sometimes called "good" cholesterol because it carries cholesterol from other parts of your body back to your liver. Your liver then removes the cholesterol from your body.

LDL stands for low-density lipoprotein. It is sometimes called "bad" cholesterol because a high LDL level leads to the buildup of plaque in your arteries.

VLDL stands for very low-density lipoprotein. Sometime VLDL is also called a "bad" cholesterol because it too contributes to the buildup of plaque in your arteries. But VLDL and LDL are different; VLDL mainly carries triglycerides and LDL mainly carries cholesterol

Triglycerides. Triglycerides are a type of fat in the blood. When you eat, your body converts calories it doesn't need into triglycerides, which are stored in fat cells. High triglyceride levels are associated with several factors, including being overweight, eating too many sweets or drinking too much alcohol, smoking, being sedentary, or having diabetes with elevated blood sugar levels.

What causes high cholesterol?

The most common cause of high cholesterol is an unhealthy lifestyle. This can include

Unhealthy eating habits,- Saturated fat, trans fat and cholesterol in the food you eat increase cholesterol levels. Try to reduce the amount of saturated fat, trans fat and cholesterol in your diet. This will help lower your blood cholesterol level. Saturated and trans fat have the most impact on blood cholesterol. Eating these fats can raise your LDL (bad) cholesterol.
Lack of physical activity, with lots of sitting and little exercise. This lowers your HDL (good) cholesterol.
Smoking, which lowers HDL cholesterol, especially in women. It also raises your LDL cholesterol.

Heredity, your age and weight may also cause people to have high cholesterol.

What kind of test measures cholesterol?

Everyone over the age of 20 should get their cholesterol levels measured at least once every five years. Your healthcare provider will order a blood test called lipid profile or lipid panel that will indicate how much cholesterol is carried in your bloodstream.

The lipid profile test gives you the following numbers:

Total cholesterol.

LDL levels.

HDL levels.

VLDL levels and triglycerides.

Non-HDL cholesterol.

Ratio between cholesterol and HDL.

Normal cholesterol level:

Men age 20 or older:

Type of Cholesterol	Healthy Level
Total Cholesterol	125 to 200mg/dL
Non-HDL	Less than 130mg/dL
LDL	Less than 100mg/dL
HDL	40mg/dL or higher

Triglyceride below 150mg/dl (normal)

Women age 20 or older:

Type of Cholesterol	Healthy Level
Total Cholesterol	125 to 200mg/dL
Non-HDL	Less than 130mg/dL
LDL	Less than 100mg/dL
HDL	50mg/dL or higher

Triglyceride below 150mg/dl (normal)

How to low cholesterol levels:

Avoid tobacco. If you do smoke, quit. Smoking is bad for you in many ways, and reducing your level of good cholesterol is one of them.
Change the way you eat. Limit the amount of trans fats and saturated fat. Eat heart-healthy foods like fruits, vegetables, poultry, fish and whole grains. Limit red meat, sugary products and dairy products made with whole milk.
Get more exercise. Try to get about 150 minutes of physical activity every week, or about 30 minutes per day for most days of the week.
Keep a healthy weight. If you need to lose weight, talk to your healthcare provider about safe ways to do this. You’ll see results even before you reach your ideal weight. Losing even 10% of your body weight makes a difference in your cholesterol levels.
Reduce the effect of negative emotions. Learn healthy ways to deal with anger, stress or other negative emotions.
Control blood sugar and blood pressure. Make sure you follow your healthcare provider’s instructions for blood sugar levels, especially if you have diabetes, and for keeping blood pressure in the healthy range.

Facts On cholesterol

1. You can’t live without cholesterol

2. 2. One out of every three adults has high cholesterol.

3. 3. High cholesterol could be genetic

4. 4. Even children can have high cholesterol.

5. 5. Sweating can raise your good cholesterol levels.

6. 6. Supplements may work to lower cholesterol — but slowly

7. 9. Women’s cholesterol levels fluctuate over their lifespan.

Top 15 immune booster foods

Immune boosting Foods

It is important to note that all these food remedies can be consumed to boost and maintain the immune system against a wide range of infections and diseases and to help manage some diseases as well as relieve certain signs and symptoms caused by diseases.

It is advisable to regularly eat these supplements to lower the risks of infections and diseases. Eat right to protect your body.

1. Almonds as Immune Booster Foods

· Lowering blood pressure

· Control blood sugar

· Regulate cholesterol level

· Ellevate constipation, respiratory disorders, and anemia.

· They have Vitamin E and C which are great immune boosters

2. Broccoli as Immune Booster Foods

· This is a vegetable that is rich in vitamins and minerals and fiber.

· They have Vitamin A, C, and E, with several antioxidants, qualifying then as one of the healthiest vegetables.

· To ensure that you get all the nutrients from broccoli, cook it for a short time or eat it raw

3. Citrus fruit as Immune Booster Foods

This is a vitamin C food that mainly helped in treating flu and colds.
They boost the immune system by increasing white blood cell production, which are the major elements involved in protecting and fighting infections in the body.
These fruits include lemons, oranges, limes, grapefruits, tangerine, and clementines.
However, it is important to consume citrus fruits daily to boost immunity continuously because the body does not produce vitamin C naturally.
They can be consumed whole or they can be squeezed into the meal while eating.

4. Elderberry as Immune Booster Foods

· Being a plant that helps in the management of flu, it may also be helpful in providing a stronger immune response against COVID-19.

· They have a laxative effect.

· They also lower sugar levels for diabetic patients.

· Generally, elderberries stimulate the immune system to produce immune elements that protect the body from foreign agents.

5. Garlic as Immune Booster Foods

· It is the most popular supplement that is well known for its ability to boost immunity.

· For centuries now, garlic has been used to manage superficial and systemic infections. It has antibacterial, antiviral and antifungal properties.

· The garlic bulbs are also rich in antioxidants that contain free radicals that can boost the treatment of Alzheimer’s, cancers and heart conditions among others. it is commonly known for its role in managing and reduction of cold and flu symptoms.

· Especially during this season of COVID-19 outbreak, garlic consumption has become an essential part of most people’s diets.

· Research has shown that people who consume garlic in their diet have fewer occurrences of cold and flu as compared to those who do not take it at all or those who use over the counter medications.

· Even so, the consumption of garlic helps shorten the period of flu and cold infections.

· It has also been documented that garlic helps reduce rates of different kinds of cancer for example, people that consume garlic have reduced cases of colorectal cancers, pancreatic and liver cancer.

· It also plays a major role in reducing blood pressure and the reduction of the hardening of arteries.

· It is also a good relief remedy for the chronic pain caused by arthritis.

· The immune-boosting properties of garlic are due to the presence of high concentrations of sulfur-containing compounds such as allicin.

6. Ginger as Immune Booster Foods

· This is a root tuber that’s known for its anti-inflammatory effect against flu and cold, especially when an individual has a sore throat and/or inflammatory illnesses.

· It also reduces nausea.

· Ginger contains gingerol which helps reduce chronic pain and also reduces cholesterol levels.

· It has several antioxidants which are very effective anti-inflammatory and immune-boosting elements. These effects are enabled by the mechanisms elicited by the body during an infection whereby, free radicals are the body causing oxidative stress. These free radicals combine with the antioxidants produced by ginger causing the anti-inflammatory effect and the immune-boosting properties.

· The anti-inflammatory effects help protect the body against arthritis, cancers, neurodegenerative disorders, and hypersensitivity.

· It has also been proven that ginger has antibacterial and antiviral effects

7. Green Tea as Immune Booster Foods

This is a herbal type of beverage packed with flavonoids.
They are natural anti-oxidants, which has several polyphenols with anti-inflammatory properties which prevents premature aging.
The antioxidants protect against cell damage which may cause chronic illnesses.
They also have a common antioxidant known as epigallocatechin gallate, EGCG which has immune-boosting properties.
Green tea has caffeine and L-theanine an amino acid that provides the calming effect, by optimizing brain function and enhance memory, elevate mood and cognitive performance.
L-theanine also helps in the production of T-cells that fight germs.
Green tea’s ability to counter oxidative stress also makes it a potent protector against neurodegenerative disease, including Alzheimer’s disease and Parkinson’s disease.
It also helps manage and maintain body weight but reducing the cholesterol content
They are great immune boosters because they have antioxidants
They have an anti-angiogenesis activity, therefore, reducing the risks of cancer and also blocking the spread of cancer cells.
They help strengthen bone density and bone development preventing occurrences of osteoarthritis and bone degenerative defects.
They help in balancing sugar levels and reduce blood sugar in diabetics.
They also maintain blood pressure and prevent heart attacks and strokes.
They protect the skin from ultraviolet rays thus reducing skin aging.

8. Kiwi as Immune Booster Foods

hey are made up of several essential nutrients such as Vitamin C, K, and Potassium. Vitamin C boosts white blood cells to fight infections and the other nutrients are essential for maintaining proper body functions.

9. Mushrooms as Immune Booster Foods

Mushrooms have high quantities of Vitamin B (riboflavin, folate, thiamin, and Niacin), Vitamin C, Vitamin D, Potassium, Magnesium, sodium, phosphorus, choline, calcium, and selenium which are very essential in boosting the immune system.
The selenium mineral has anticancer, antioxidant and anti-inflammatory properties. Therefore, the presence of selenium in mushrooms is an implication of its importance in reducing cancer such as lung cancer, prostate cancer, and breast cancer according to the National Cancer Institute.
Selenium also reduces and prevents heart disease risks, and thyroid disease. However, more research and trials are being done to confirm these properties.
Choline is also an antioxidant known to reduce risks of prostate cancer, studies have proven.
The mushrooms are well known for their immune-boosting properties. They have a special amino acid known as ergothioneine, a very powerful antioxidant which provides protection from free radical during infections and they also boost immunity.
Some mushrooms have natural antibiotics that inhibit microbial growth and some fungal infections. these naturally produced antimicrobial properties also boost immunity by stimulating and regulating its responses.
Mushrooms are complex carbohydrates (Polysaccharides) which also have an immune-boosting effect.
Other important properties of mushrooms include:

lowering cholesterol levels by using its fiber and enzymes to balance cholesterol levels.
they are low energy and low carbohydrate foods which are ideal for a diabetic individual diet
they have high quantities of calcium which help in bone growth and strengthening preventing bone defects like osteoporosis and bone degradation.
presence of vitamin D help in the absorption of calcium and phosphorus which are very essential to the body.
presence of antimicrobial agents naturally produces by mushrooms helps in the healing of stomach ulcers and wounds.
they have potassium which is a natural vasodilator thus it plays a primary role in lowering blood pressure hence reducing conditions of heart attacks and stroke.
mushrooms are also good supplements of folate which are required by pregnant women to boost fetal health.

10. Papaya as Immune Booster Foods

This is a nutritious fruit that is made up of large quantities of Vitamin C.
It has digestive enzymes called papain which have anti-inflammatory effects.
Papain also acts as a laxative, reducing constipation.
They also have potassium, Vitamin B, and folate which have overall positive health properties.

11. Pomegranate and pomegranate juice as Immune Booster Foods

Pomegranate seeds have a bright red color acquired from polyphenols, a powerful antioxidant that is at 3 times higher as compared to other fruits even red wine and green tea. The antioxidants help in the removal of free radicals which may cause cell damage and reduce inflammations.
Compounds produced bt pomegranates have an active antibacterial activity against E.coli, Yersinia, salmonella, shigella, Clostridium, Staphylococcus aureus, Listeria. They inhibit bacterial growth in the mouth which may cause periodontal disease, plaques, and gingivitis.
They also have antiviral activity against viral flu and herpes.
They also promote the growth of beneficial gut flora that boosts immunity such as Lactobacillus and Bifidobacterium.
They are rich in Vitamin C with a single pomegranate fruit possessing about 40% of Vitamin C content a day. Making juice from the pomegranate seeds breaks down Vitamin C providing more nutrients.
Preliminary research recently showed that pomegranate juice helps to stop the growth of prostate cancer cells and reduce the risks of cancers.
The antioxidants in the pomegranate juice have antioxidants that slow down the progression of Alzheimer’s and protect memory.
They reduce gut inflammation and improve digestion therefore it is beneficial to people with Crohn’s diseases, ulcerative colitis, and bowel disease. In case of an allergic reaction that causes diarrhea, avoid taking the fruit until the bowel movement stops.
Flavonols found in pomegranate juice blocks inflammations that cause and progress osteoarthritis and cartilage damage. More studies are being conducted in regard to its role in osteoporosis arthritis and other joint inflammations.
This is a healthy heart fruit which protects the heart and its arteries. a few studies have shown that pomegranate juice improves blood flow and prevents the hardening of the arteries. It also reduces the accumulation of cholesterol in the arteries. But note that it can affect blood pressure and can react to cholesterol medication.
Pomegranate juice reduces systolic blood pressure which promotes heart health and reduces blood pressure.
They are a very good source of folate, potassium and Vitamin K.
It is also a memory booster, at least intake of 8 ounces of pomegranate juice daily improves learning and memory.
It also reduces risks of infertility in women and increases levels of testosterone in men and women.
It is also used as a remedy for diabetes by decreasing insulin resistance and lowering blood sugar.
Pomegranate can be consumed by blending juices, added to lemon juices, adding color to fruit salads and to make meat tasty.

12. Red bell peppers as Immune Booster Foods

This pepper has twice the amount of vitamin C the number of citrus fruits.
They also have beta carotene are also very efficient immune boosters.
In addition to boosting your immunity, red pepper also plays a role in maintaining healthy skin and eyesight.

13. Spinach as Immune Booster Foods

This is one of the richest green vegetables, otherwise known as a superfood, known for its richness in Vitamin A, C, fiber, beta carotenes, Magnesium, calcium, iron and several antioxidants which are properties that increase the body’s ability to fight infections.
It is a dark, leafy green vegetable with benefits of skin, hair and bone health.
It is healthy when cooked in low/medium heat (lightly) which allows the release of Vitamin a and oxalic acids and to preserve the vegetable nutrients.
They are also essential nutrients to mediate cell division processes and DNA repair.
it contains antioxidants such as alpha-lipoic acid which lowers glucose levels, increasing the sensitivity of insulin and prevents oxidative stress caused by the effects of diabetes.
Alpha-lipoic acid also decreases peripheral neuropathy in diabetics. Alpha-lipoic acid can be prepared as an oral supplement with the same benefits.
when consumed raw or lightly cooked, it preserves the chlorophyll pigments, whose benefits have been studied in animals and one of them being the blocking/prevention of the carcinogenic effect of heterocyclic amines.
presence of the beta-carotenes reduces the risks of asthma, especially in children.
the combination of iron and vitamin K strengthens bone preventing bone fracturing and bone defects.
spinach has enough fiber (roughage) quantities that prevent constipation.
vitamin A produced by spinach prevents acne and skin abrasion.
Vitamin C also contributes to the production of collagen that is essential in strengthening hair and also hair growth and healing of skin abrasions.
iron is important in the production of red blood cells maintaining blood levels preventing the development of anemias.

14. Turmeric as Immune Booster Foods

Curcumin is the main active ingredient in turmeric with powerful anti-inflammatory properties, which blocks inflammation at the molecular level.
Curcumin has also boosted brain development, improved brain function and reduces the risks of brain disease.
They are also very strong antioxidants which reduce skin aging and prevention of diseases by blocking free radicals.
They have high quantities of curcumin which contributes to decreasing exercise-induced muscle damage.
It is used to relieve the pain caused by osteoarthritis and rheumatoid arthritis.

15. Yogurt as Immune Booster Foods

Good and beneficial yogurt has a live and active culture that stimulates the immune system to help fight diseases. It is a fermented dairy product prepared from milk with added probiotics and sugars or/and flavors bacteria. During bacterial fermentation, lactic acid is produced which makes milk proteins to curdle producing a unique texture and flavor.
Having been consumed for hundreds of years, its been proven to be very nutritious, and a great immune booster that fights diseases.
It is a great source of Vitamin D, which regulates the immune system and boosts natural defense mechanisms against diseases.
They can be taken unsweetened or flavored. Unsweetened yogurt is the best which can be mixed with fruits and honey to test. Plain yogurt without added colorants is a white, thick liquid with a tangy flavor.

they contain calcium, a very essential mineral for growth of teeth, and bones. A cup of yogurt offers at least 49% of calcium.
It is also high in B vitamins, particularly vitamin B12 and riboflavin, both of which may protect against heart disease and certain neural tube birth defects.
A daily dose of yogurt gives 38% phosphorus, 12% for magnesium and 18% for potassium. These minerals regulate blood pressure, bone health, and body metabolism.
They have fortified Vitamin D which promotes bone and immune system health reducing risks of diseases like heart disease and depression.
Yogurt contains about 200grams of proteins which play a major role in increasing energy metabolism. they also regulate appetite and delay hunger as they also increase hormone production.
Some yogurts have live bacteria (Bifidobacteria) or probiotics which contribute greatly to reduce Irritable Bowel Syndrome (IBS) a disorder of the large intestines. They also reduce bloating.
Yogurt probiotics, vitamins, and minerals are great immune boosters that prevent certain illnesses.
They also have phosphorus and Vitamin D, which increase bone density, reducing the risks of osteoporosis and healing of bone fractures.
It also helps in weight management by increasing appetite and reducing hormone production for YY and GLP-1ogurt has several properties that may help with weight management. Consumption of yogurt is linked to lower body weight, less fat percentage and waist circumference.
It also reduces the risks of obesity development because of low-calorie levels.

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