i want 25 mutiple choice questions with their model answer below each question about all of the following indormation :Contents
• PCR concept.
• Applications of PCR.
• PCR reaction components.
• PCR cycle.
•Types of PCR.
• polymerase chain reaction (PCR) is an in-vitro enzymatic DNA amplification technique for amplification of a region of DNA.
• The amplified DNA sequence must be known or it lies between two regions of known sequence.
Introduction
⦿PCR allows the synthesis of millions of copies of a specific nucleotide sequence in a few hours.
⦿ DNA can be obtained from any source(viral, bacterial, plant, animal)
.1Genome mapping and gene function determination.
2. Biodiversity studies ( e.g. evolution studies).
3. Detection of drug resistance genes.
4. Forensic analysis of DNA samples.
5. DNA fingerprinting for analysis of evidence from crime scenes using a single hair or spot of blood.
• Prenatal testing of genetic diseases
• Early detection of cancer (for example, screening of family members in case of genetic types of cancer.
• Diagnosis of viral infections (Hepatitis B, C, AIDS, COVID19,…ETC)
• Diagnosis of genetic disorders: sickle cell anemia,…etc
6. Diagnostics:
Template DNA: a sample of the DNA that you wish to replicate.
Two Primers (forward and reverse):
Are starting/ending points for DNA replication; thus define the region of DNA to be replicated in PCR.
Primers range from 15 to 30 nucleotides, Synthetically produced, complimentary to the 3’ ends of target DNA, not complimentary to each other).
• DNA polymerase: (Enzyme that replicates
DNA). The most commonly used is the
Taq DNA polymerase (Thermostable DNA
polymerase).
• dNTPs (DeoxyribonucleosideTriphosphates)
(dATP, dCTP, dGTP, dTTP).
• Mg2+ : Essential co-factor of DNA polymerase.
1. Denaturation of DNA
The target DNA to be amplified, is heated to 95°C to separate dsDNAto single strands.
2. Annealing of Primers
A Primer is a single stranded oligonucleotide molecule, complementary to sequences surrounding the target sequence. Two primers are required for PCR reactions.
During the annealing phase, DNA is cooled to 50°C& the 2 primers (one for each strand) anneal to complementary sequence on the ssDNA.
3. Extension of Primers
DNA polymerase uses dNTPs in excess to initiate the synthesis of 2 strands complementary to the original ones. The temperature is adjusted to 72°C in this phase.
A programmable device that cycles between specific temperatures for set periods of time – used for PCR.
⦿ 20-30 cycles are run, amplifying DNA by a millionbillion fold “exponential amplification”.
⦿ A special DNA polymerase must be used that can withstand high temperatures of the reaction. Thermus acquaticus bacterial Taq Polymerase.
⦿ Resulting PCR products are separated by gel 
electrophoresis transilluminator.	& 	visualized 	by 	UV 
Different Types Of PCR
CONVENTIONAL PCR
Normal PCR process. Here the primers bind specifically to each of 2 DNA strands.
All that needed for the PCR process are PCR tubes, DNA polymerase, buffer, primers and target DNA.
►The whole process takes place within 35-40 minutes repeatedly and viewed by gel electrophoresis technique.
Multiplex-PCR:
It is a special type of the PCR used for detection of multiple pathogens by using Multiple primers sets each one targets a particular pathogen.
Uses:
This permits the simultaneous analysis of multiple targets in a single sample.
Nested-PCR:
Used to increase the specificity of DNA amplification.
Two sets of primers are used in two successive reactions.
In the first PCR, one pair of primers is used to generate DNA products, which will be the target for the second reaction.
Using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set, thus increasing specificity.
Uses:
Detection of pathogens that occur with very few amount.
RT-PCR (Reverse Transcription PCR, Real Time-PCR)
Used to reverse-transcribe and amplify RNA to CDNA.
PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. Complementary
The two reactions may be combined in a tube.
Uses:
Hepatitis & Viraj
1-Detection of RNA virus like (HCV).
Quantitative Real-Time PCR (qRT-PCR)
Method use fluorescent dyes, such as Sybr Green, or fluorescence-containing DNA probes, such as TaqMan, to measure the amount of amplified product as the amplification progresses.
Quantitative - PCR:
Used to measure the specific amount of target DNA (or RNA) in a sample.
By measuring amplification only within the phase of true exponential increase, the amount of measured product more accurately reflects the initial amount of target.
Special thermal cyclers are used that monitor the amount of product during the amplification.