Hey all. I sincerely apologise for the late posting as I was confused over the dates and the weeks. Anyway, I’ll continue from where I left of the previous post that is gel check for primer optimization. After the gel check for primer optimization and the optimum conditions have been chosen for the specific primer, PCR can then be done for that primer.
What are the differences and similarities between primer optimization and PCR?
Differences
-Primer optimization is done using test DNA whereas PCR is done using a specific patient’s (whether healthy or diseased) DNA. This is because, when doing the primer optimization, the optimum conditions are not known, thus, it is not necessary to use patient’s DNA. (Patient’s DNA is very precious as they are retrieved only ONCE from the patient themselves while test DNA can be bought.)
-For primer optimization, a range of temperatures will be programmed at the Thermogradient, whereas, for PCR, a specific optimum temperature will be set for the Thermocycler.
Similarities
- The method for conducting both PCR and primer optimization is similar including all the reagent used.
Introduction on PCR
Polymerase Chain Reaction (PCR) is a method for the amplification of specific DNA sequences in vitro, which involves repetitive heating and cooling cycles to yield large quantities of replicated DNA. It requires two oligonucleotide primers that will flank the DNA target sequence that is to be replicated. The three major steps that are carried out in each PCR cycle are the following:
1. Denaturation: The hydrogen bonding that holds the double stranded of the target DNA molecule together, are separated into single stranded DNA by heating at 940C.
2. Annealing: The two single stranded DNA are then allowed to cool, at the temperature ranging from 45oC to 60oC, where annealing of the primers to the single stranded DNA will take place. This would initiate the synthesis of the complementary sequences.
3. Extension: When the temperature is increased to a temperature optimum for the Taq DNA polymerase, it will bind to the free 3’ end of the primers. By incorporating dNTPs, the Taq DNA polymerase synthesizes a new DNA strand in a 5’ to 3’ direction.
Method for PCR
*Please refer to previous entries on method for primer optimization as they are similar*
After which, gel check has to be done again. This time, it is to double check if the bands are present for different DNA samples. However, for the gel check of PCR, only 0.5ul of loading dye and 2ul of random PCR product are used instead of 2ul of loading dye and 10ul of primer optimization product. This is because, the remaining 8ul of the PCR product is required to be used for experiments following this step. If approximately 80% of the bands appear, purification of the PCR product can be done.
Purification
To eliminate excess PCR components after the PCR reaction, purification of the amplified PCR products was done using two hydrolytic enzymes, Exonuclease 1 (Exo 1) and Shrimp Alkaline Phosphatase (SAP). Exonuclease 1 catalyzes the removal of excess primers in the 3’ to 5’ direction while Shrimp Alkaline Phosphatase is responsible for the dephosphorylation of dNTPs to prevent it from participating in any further polymerisation steps. Both enzymes at its optimum temperature, 37oC, are involved in the digestion process. Following this, the temperature will increase to 72oC resulting in the denaturation and inactivation of the enzymes. This would prevent the enzymes from interfering in subsequent steps, for example, cycle sequencing.
Methods for purification
Following PCR and gel electrophoresis, 0.5µl of Exonuclease 1 (Exo1) and 1µl of Shrimp Alkaline Phosphatase (SAP) need to be added into each sample of the PCR products.
1. Calculations were done by multiplying the number of samples needed to be purified.
2. One 1.5ml of eppendorf tube (master mix tube) was collected and labeled ‘Exo-Sap’.
3. An ice box, filled with crushed ice, was prepared.
4. Exo 1 and SAP was retrieved from -20oC fridge and placed in the ice box.
5. The desired amount of Exo and SAP was pipetted into the master mix tube.
6. The master mix tube was then spun down using a mini centrifuge.
7. 1.5ul of the resultant mixture was pipetted into each sample in each well.
8. The samples of the PCR products were placed into the thermocycler and the program was set.
After purification is done, the next step would be sequencing. I would explain this step in further details in the next posting. Till then, I would like to wish all Muslims, Selamat Hari Raya Aidilfitri and to all non-Muslims, have a joyous holiday! J
Liyanah Zaffre
0607718D
TGO2
Week 14 posting
Answers for WEEK 13
As most of the questions asked are pretty similar and pictures are requested, this post will show some of the common casts and crystals that I've encountered in my lab. The following pictures are retrieved from websites since I wasn't allowed to snap photos in my lab. They're actually quite easy to find on the net.
1) Waxy casts
I was told that waxy casts normally appear as the end stage of casts formation. It basically means, a waxy cast appears when the patient has been suffering from renal failure for a long time. It is originally a normal cast but appears as waxy after years of suffering from renal failure. It normally appears in diseased patients. So far, I've only encountered one throughout my whole week stay under urinalysis. They are normally broad with sharp edges and their ends looked as if it has been cut or broken. They are also more distinct than hyaline casts.
Retrieved 28th September 2008 from, http://www.academic.marist.edu/~jzmz/methods/waxycast.jpg
2)Granular casts
Granular casts are quite normal and may be due to breakdown of other cellular casts such as RBC casts or leukocyte casts. Sometimes, their appearance may indicate chronic renal failure but similar to hyaline casts, they may appear due to stress or after strenuous exercise and may disappear after sometime. Notice the granules found on the cast, making it look like a big clump of granules.
Retrieved 28th September 2008 from, http://www.agora.crosemont.qc.ca/urinesediments/image/d21i003.jpg
3) Hyaline casts
Hyaline casts may be quite tricky to identify due to its faint appearance and almost blends in with the background. Therefore, as I've mentioned in my earlier post, casts are usually looked out for when protein levels are high. They appear in urine due to solidification of Tamm-Horsfall mucoprotein secreted by tubule cells. They are more common in acidic urine. Patients who have done vigorous exercises or are feeling dehydrated, may have hyaline casts in their urine. The arrows pointing in the picture are trying to show the smooth edges of the hyaline cast.
Retrieved 28th September 2008 from, http://www.med.uiuc.edu/internalMed/residency/edmod/mod1/hyaline.jpg
4) Calcium Oxalate Crystals
Calcium Oxalates are very easy to distinguish due to its "square" shape and crosses found in the middle. A senior colleague told me that sometimes, presence of calcium oxalates may be due to patients eating too much vegetables. These type of crystals are quite common. They do not necessarily indicate any diseased conditions. They may or may not develop into kidney stones depending on how hydrated the body is.
Retrieved 28th September 2008 from, http://library.med.utah.edu/WebPath/jpeg2/URIN077.jpg
5) Triple Phosphates
This is another type of common crystals found in normal patients. To me, they look like gold bars, though some people would prefer to think of them looking like coffin lids. They don't have crosses in the middle like calcium oxalates. They appear due to ammonia concentrations found in the urine making it slightly alkaline. High amounts may indicate urinary tract infection. But very small amounts may not be clinically significant and does not indicate any diseased state.
Retrieved 28th September 2008 from,
http://meded.ucsd.edu/isp/1994/im-quiz/images/struvit.jpg
6) Uric Acid
As I've mentioned, these crystals are quite normal and are usually found in urine with pH 5 or lower. The rossette form of uric acid is the more common form. It is basically the flower-shaped ones. Uric acid may accumulate and are formed due to breakdown of purines or waste products in the body. Too much uric acid may cause gout to develop. If they accumulate in the kidneys, kidneys stones may be formed.
Retrieved 28th September 2008 from, http://www.academic.marist.edu/~jzmz/methods/uacystals.jpg
These are the common crystals and casts that I've encountered so far. I'm sure there are more but are less common in patients. The results printed by the analyzer would usually show the amounts of the substances present. Usually, plus signs '+' are used to indicate the levels of the substances present. For example, 'Leukocytes ++++' means about 30-50 leukocytes are present. We would then report the results into the system as Leukocytes 4+. The system would automatically know that there are 30-50 leukocytes detected.
We usually spin the urine samples if high amounts of leukocytes are detected or when high amounts of erythrocytes are detected or both. We also spin turbid or slightly turbid samples. I was told that if the urine is not spun down, we might miss out certain substances that are present in the urine. Therefore, by spinning them down and allowing the substances to settle at the bottom and then examining the sediment, would enable us to identify almost all the substances that are present in the urine sample. If proteins appear to be 3+ or 4+, we also spin the urine sample down to observe for any casts.
The doctors who ordered for this test would usually simply indicate UAN or URINE FEME on the request form. Both would mean that urinalysis and urine microscopy is required to be done upon the urine sample.
Urine Specific Gravity is an indication of how concentrated or how diluted the urine sample is. The more concentrated the urine, the higher the specific gravity is. The values are usually fixed like 1.000, 1.010, 1.015, 1.020, 1.0250, 1.0300 and so on. Increased specific gravity may be due to dehydration or increased secretion of anti-diuretic hormone in the body. Decreased specific gravity may indicate damage to the tubule cells causing the kidneys to be unable to reabsorp water, kidneys producing too much urine or renal failure. Specific gravity of 1.002 to 1.035 is considered normal.
I hope this second posting is much more clearer to all of you and I hope I've managed to answer all the queries posted earlier. Thank you C:
Nur Azeimah
0607060A
TG 02
