Transcript
PCR-RFLP Report Polymerase Chain Reaction (PCR) The Polymerase Chain Reaction is a method that uses the capability of DNA polymerase to synthesize to new DNA strands which are matching to the template strand. A primer needs to be added to the first nucleotide due to the fact that DNA polymerase only can add a nucleotide only onto a 3'-OH group that already exists. Because of this condition, we are able to define a chosen region of template sequence which we can then generate millions to billions of copies. This technique was developed by Kary Mullis in 1983 and is a very common indispensable technique which has a variety of uses such as DNA cloning for sequencing, genetic fingerprints, detection and diagnosis of infectious disease (often cancer), etc.
Figure 1
Procedure: PCR consist of 20-40 cycles of repeated temperature change and each cycle has mostly 3 distinct temperature steps. The temperature used and the duration of treatment in each cycle depends upon many factors such as melting temperature of the primers, etc. The cycle usually starts by a high temperature of greater than 90 degree Celsius and ends with the same treatment as the initial step. Starting Step: The reaction is heated to a temperature between 94-96 Degrees Celsius or 98 Degrees Celsius if polymerase which can withstand higher temperatures. This continues for 1-9 minutes. (This step is only required of the DNA polymerase requires heat activation) Denaturing Step: This is the first step of the cycle. This step requires melting of the DNA template by disturbance of hydrogen bonds between complementary bases which the resultant being a single-strand of DNA molecules. To achieve this, the reaction must be heated to 94-98 Degrees Celsius for about 20 to 30 seconds.
Annealing Steps: Annealing must take place in order for the hybridization of primers to the single stranded DNA template. This temperature must be high enough in order for the hybridizations to bind specifically and low enough that it is possible for hybridization of primers to occur. Annealing temperature is usually 3-5 Degrees Celsius lower than the melting temperature of the primers. Extension/elongation step: This step consists of DNA polymerase synthesizing with a new DNA strand which is complementary to the DNA template strand. This is done by adding dNTP's which complement the template in the 5'-3' strand, condensing the 5'-Phosphate group of the dNTP along with the 3'hydroxyl group at the end of the nascent extending DNA strand. The temperature at which the reaction is treated depends upon the primer used. The duration of this step also depends on the DNA polymerase used also, and the length of DNA fragment used. Under optimal required temperature of the primer, the DNA polymerase will polymerize a thousand base pairs per minute. Final elongation: After the last PCR cycle is completed, the reaction is cooled to a temperature of 70-74 Degrees Celsius (optimal temperature needed for activity for most polymerases used in PCR) for 5-15 minutes to ensure that remaining single-stranded DNA is fully exte nded. Final Hold (optional): Cooled to a temperature of 4-15 Degrees Celsius for short term storage of the reaction. To see if PCR has successfully generated expected DNA fragments, Agarose Gel Electrophoresis is used for size separation of the PCR products.
Restriction Fragment Polymorphism (RFLP)
Length
Restriction Fragment Length Polymorphism (RFLP) is a technique that uses the difference in homologous DNA sequences that can be detected the presence of fragments which are of different lengths after digestion of the DNA samples. Because this method acts as a molecular marker, RFLP is specific to a combination of clone/restriction enzyme. Double stranded DNA is cleaved by hydrolyzing the support which is between the Deoxyribose sugar and the phosphate the 5' phosphate and the 3'OH, this is done by the restriction enzymes. Loosely bound initially, these enzymes bind to the DNA and move along the path until a recognition sequence has come in contact. symmetrical The
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enzyme is able to cleave the DNA when the tighter binding with the recognition sequence leads to a symmetrical change. Because there are many polymorphisms in the genome, it is possible that some of the variations end up occurring at the recognition sequences for restriction enzymes. If a sequence such as GAATTC would normally be recognized by an enzyme, or conversely, a non-cleavage sequence could be changed to a recognition sequence, making the enzyme cut the DNA. The different cleavage sites lead to differences in length of the DNA segments after digestion. Digested DNA can display a detectable difference in bands from two individuals if it is run through gel electrophoresis. This information can then be used in a wide variety of situations.
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Question: What causes Sickle Cell Anemia?
Sickle Cell anemia is a common type of sickle cell disease which makes the body produce red blood cells in the shape of a sickle, "Sickle shape " intimates that the red blood cells are molded like a bow. Ordinary red blood cells molded and look like disks and are more effective in moving through the veins. Red blood cells contain an iron-rich protein called hemoglobin. This protein carries the oxygen and delivers it to any part of the body. However, Sickle cells contain bizarre hemoglobin called sickle hemoglobin or hemoglobin S which is the cause to its peculiar shape. Sickle cells are firm and sticky and have a tendency to stop circulation system in the veins of the organs and limbs. Blocked blood stream can result in excruciating pain and correspondingly can cause organ harm. It likewise raises the danger of getting an infection.
Sickle cell is the effect of a change in the #6 amino acid of the ß-globin chain of hemoglobin. Particularly glutamic acid is changed over to valine, As seen in Figure 5, the gene of an individual with a normal genome (because large percent of the human genetic code is similar to each other, so we can identify which letter in the code is abnormal) compared to another individual with Sickle Cell Anemia. This results from a change in the nucleotide A to T. This change kills a site perceived by the limitation compound DdeI and is called a missense-severe mutation .The restriction enzyme in this would be Ddel (sequence: 5'-GTNAG-3') and the probe will be a fragment ß- globin coding sequence. Using RFLP, we can identify the mutation in genetic code. Figure 4 Because Sickle Cell Anemia is a genetic disorder which relies only on one letter of the sequence, the procedure is simplified as we only need to test the beta-globin gene. When both the DNA strands are compared, the bar for 6th amino acid will have a different blot showing. Hence, we can Figure 5 conclude that Sickle Cell Anemia is caused by a missense-severe mutation in the 6th amino acid of the ß-globin chain of hemoglobin.
Biography http://www.ncbi.nlm.nih.gov/probe/docs/techpcr/ http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/ http://en.wikipedia.org/wiki/Polymerase_chain_reaction *(http://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism#Analysis_technique) http://www.nhlbi.nih.gov/health/health-topics/topics/sca/ http://upload.wikimedia.org/wikipedia/commons/9/96/Polymerase_chain_reaction.svg http://www.ndsu.edu/pubweb/~mcclean/plsc431/markers/marker1.htm Images http://openwetware.org/images/3/35/Pierce_18_19_large_2.jpg (Figure 1) http://homepage.smc.edu/hgp/images/rflp.gif (Figure 2) http://upload.wikimedia.org/wikipedia/commons/d/d0/Roland_Gel.JPG (Figure 3) http://www.nhlbi.nih.gov/health/health-topics/images/sickle_cell_01.jpg (Figure 4) http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SickleMutation.gif (Figure 5)
