Osama Nabil Qasem
1. What are the principles of PCR? The polymerase chain reaction (PCR) is a Biochemical technology Simulate DNA replication in vivo , which the DNA polymerase use to amplify a specific DNA sequence by elongation primer through out adding dNTPs . [1]
2. What is important when you design primers? there are several considerations for primers design including primer length between 18 and 24 bases, Melting Temperature (Tm) , 3' sequence and guanine-cytosine content . [2]
3. Describe how PCR is performed! ● step 1- DNA , tow primer (forward and reverse primer), dNTPs , and DNA polymerase enzyme mixed together in sutable tube. [1]
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[1] ●step 3- decrease temperature to anneal primer on specific DNA sequence, which is annealing temperature depends on melting temperature Tm of primer. [1] ●step 4- rise the temperature to 72 °C is optimal degree for DNA polymerase enzyme to extend complementary specific DNA sequence. [1]
4. Does PCR have any limitations/advantages? Advantage : PCR produces a billions of copies of a specific DNA sequence, which allows detection and identification of gene sequences using visual techniques based on size and charge. through out PCR technology can applied on study gene , diagnosis of genetic and infectious diseases Forensic applications.[3]
Limitation : The specificity to produce new DNA strands and yelled may be altered by nonspecific binding of the primers to other similar sequences on the template DNA or by sample contamination.[3]
5. Describe and explain what the following types of PCR are: ● DOP-PCR is new method used to amplify sufficient amounts of DNA from small amounts of
It helps medics to find a direct genetic cause of the patient’s condition and target it with pharmaceutical or other therapies. The technology is used for the identification of DNA sequences that increase risks of current diseases and disorders; with this information carriers can start to make efforts to prevent them before the development of the problem. The video mentioned 200 actionable genes, structures that have direct links with a specific condition. Knowing about their presence, people have a chance to bring in preventive measures like taking anticoagulants in the case of identification of a thrombogenic gene. The technology led to the significant improvement of diagnostics and personalized treatments. It helped to find a rare, life-threatening mutation in case of Beery twins and assign a drug to a girl (Alexis) that returned her to a normal life. In the case of cancer genome sequencing led to the development of genetic drags, which target essential tumor genes and make malign structures to shrink. The video mentioned a product that works with the BRАF protein that induces cells to uncontrolled division; the drug led to the remission in the patient with metastasizing melanoma. Such treatment was effective in the case of cystic fibrosis. In the case of the breast cancer the technology helps to evaluate the aggressiveness of the condition and make a personalized decision about chemotherapy. The video also mentioned the pre-implantation genetic diagnosis – an early-staged technology that prevents the development of inherited disorders in
Using PCR and Gel Electrophoresis to Determine Genotype. In certain situations, it is necessary to identify DNA retrieved from a sample. When there is a small sample in need of identification, Polymerase Chain Reactions are used to multiply the DNA. in the sample in many identical samples.
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
PCR or polymerase chain reaction is not a DNA typing technique, but a variety of different DNA tests (Riley). PCR duplicates and increases the quantity of a DNA strand which is beneficial to forensic scientists who are faced with little quantity of materials (Saferstein 394). The introduction of PCR-based testing in DNA analysis required scientists to switch to smaller targets that had the same repetitive variation (Jones). This is how short tandem repeat, the newest method of DNA typing,
...oligomer, possible truncated failure sequences with free 5’hydroxyl ends, byproducts of deprotection, and silicates from hydrolysis of the glass support. Different purification methods can be used to separate the product oligonucleotide from the contaminating species.
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
Once a crime has been committed the most important item to recover is any type of evidence left at the scene. If the suspect left any Deoxyribonucleic acid (DNA) at the crime scene, he could then be linked to the crime and eventually charged. A suspect’s DNA can be recovered if the suspect leaves a sample of his or her DNA at the crime scene. However, this method was not always used to track down a suspect. Not too long ago, detectives used to use bite marks, blood stain detection, blood grouping as the primary tool to identify a suspect. DNA can be left or collected from the hair, saliva, blood, mucus, semen, urine, fecal matter, and even the bones. DNA analysis has been the most recent technique employed by the forensic science community to identify a suspect or victim since the use of fingerprinting. Moreover, since the introduction of this new technique it has been a la...
Lyons, R.H. (2004). How do we Sequence DNA? In A Primer in DNA Structure and Function. Retrieved from http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
"Polymerase Chain Reaction (PCR) Fact Sheet." National Human Genome Research Institute. 10 Dec. 2007. National Institutes of Health. .
The age of genetic technology has arrived. Thanks to genetic technological advancements, medical practitioners, with the help of genetic profiling, will be able to better diagnose patients and design individual tailored treatments; doctors will be able to discern which medications and treatments will be most beneficial and produce the fewest adverse side effects. Rationally designed vaccines have been created to provide optimal protection against infections. Food scientists have hopes of genetically altering crops to increase food production, and therefore mitigate global hunger. Law enforcement officers find that their job is made easier through the advancement of forensics; forensics is yet another contribution of genetic technology. Doctors have the ability to identify “high-risk” babies before they are born, which enables them to be better prepared in the delivery room. Additionally, oncologists are able to improve survival rates of cancer patients by administering genetically engineered changes in malignant tumors; these changes result in an increased immune response by the individual. With more than fifty years of research, and billions of dollars, scientists have uncovered methods to improve and prolong human life and the possibilities offered by gene therapy and genetic technology are increasing daily.
Then the sequence was loaded into Velvet where it was trimmed to the desired k-mer length for alignment and contig formation. Mitos and MEGA alignment Explorer were also used in order to get the DNA sequence to a
Gel electrophoresis is used in a variety of settings, particularly in molecular biology. Besides being used to separate nucleic acids, such as DNA and RNA, gel electrophoresis is also employed to divide proteins (Gel Electrophoresis). According to research, electrophoresis is applied for the following reasons, "To get a DNA fingerprint for forensic pur...
scientific method for things such as DNA samples from hair and body fluids, fingerprint analyses and
The scientific and medical progress of DNA as been emense, from involving the identification of our genes that trigger major diseases or the creation and manufacture of drugs to treat these diseases. DNA has many significant uses to society, health and culture of today. One important area of DNA research is that used for genetic and medical research. Our abi...
Step 1 is repeated by using different yeast strains, a pet 1 and M240 into all 6 conical