Hey all. Time sure flies and its already our last week of SIP next week! Anyway, I will continue from where i left the previous entry.
After purification is done, the sequence of the individuals DNA need to be determined by carrying out sequencing.
DNA sequencing using dideoxy-chain termination method or Sanger sequencing is a technique used to determine the exact nucleotide sequence of a gene, which includes adenine, guanine, cytosine and thymine. It uses DNA synthesis as the basis for the sequencing reaction and also incorporates dideoxynucleotide triphosphate (ddNTPs) as chain terminators. Firstly, the DNA template is denatured and primer is allowed to anneal. DNA polymerase will synthesize the complementary strand by incorporating the dNTPs, in which the strand will only terminate once a ddNTP that lacked a 3’ hydroxyl group is incorporated. After sequencing, the strands of different lengths are separated by capillary gel electrophoresis. As the fragments migrate out of the gel, they are hit by a laser beam, causing the ddNTPs to fluoresce. The data is converted to an electropherogram for visualization.
In sequencing, there are 2 major parts to it that is 1) Cycle Sequencing 2) Ethanol precipitation.
Cycle sequencing is carried out in three major steps, similar to PCR except one primer is used in the reaction.
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. However, only one primer, either forward or reverse will be used in cycle sequencing.
3. Extension: In this step, Taq polymerase will bind to the DNA, allowing the synthesis of complementary strand by incorporating dNTPs. When a dye-labelled ddNTP is incorporated, it cannot form a phosphodiester bond with a normal nucleotide, thus, terminates the synthesis of the new strand.
This reaction was carried out using the Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The components listed below will be added to the sequencing plates for sequencing. The reaction was terminated using equal volumes of stock solutions consisting of 3M sodium acetate and 125mM EDTA, after cycle sequencing.
Components of Cycle Sequencing Reaction
To precipitate out the DNA, ethanol precipitation was done by adding 25µl of 100% ethanol to the PCR products in the sequencing plates. It will then be incubated for 15 minutes by wrapping the plates with aluminium foil. After which, the plate was centrifuged at 3000G, 4oC for 30 minutes to obtain the DNA pellet. To remove the 100% ethanol that is the supernatant, the plate was inverted on a blotting paper and pulsed at 200G for 20 seconds. Following this, to wash the DNA pellet, 70µl of 70% ethanol will be added and again, the plate was centrifuged at 1700G, 4oC for 15 minutes. Subsequently, it will be pulsed twice at 200G for 20 seconds to remove the 70% ethanol. To properly ensure that the pellet was completely dried, the plate was placed in the vacuum dryer for 5 minutes followed by 95oC for 5 minutes in the opened-lid thermocycler. 20µl of formamide was then added to dissolve the DNA pellet before it was placed inside the 3730 DNA Analyzer (Applied Biosystems, USA) to be sequenced.
After sequencing is done, the results will then be produced as trace files by the DNA analyzer machine and loaded into a computer program for blasting and genotyping to be done.
Blasting is a method used to locate single nucleotide polymorphisms in the fragment. A software will then align the trace files, in which all the different DNA sequences of each individual DNA will be aligned accordingly. After which, the DNA sequences will be compared and see if there is the presence of double peaks or change in the colour of peak at the same location. This is a high indicative of a SNP. After blasting is done, and the SNPs had been located, genotyping can then be done for each individual.
Using Sequencing Analysis v5.2 (Applied Biosystems, USA) and SeqScape v2.5 (Applied Biosystems, USA), the resultant trace files from the DNA sequencing reactions were base called and assembled respectively. Following assembly, analysis of the DNA sequences was then done by multiple sequence alignment and comparing to the reference obtained from National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) to detect single nucleotide polymorphisms. Each individual will be either having genotypes either 1) homozygous wild type, 2)heterozygous 3)homozygous variant.
For example, for SNP A>G,
Homozygous wild type : AA
Homozygous variant: GG
Hey all. Time sure flies and its already our last week of SIP next week! Anyway, I will continue from where i left the previous entry.
So, this week I would lke to share with you another analyzer called the Elecsys 2010 by Roche Diagnostics, USA. Elecsys 2010 is used to perform immunological tests, mainly in vitro testings for the quantitative and qualitative determination of analytes present in the human serum.
The kind of immunoassays carried out in my lab are tumor markers such as CA 125 and CA 19-9, Free T4 and Free T3, Carcinoembryonic antigen (CEA), Hep-B surface antigen type II, Hep-A virus, HIV, Thyroid Stimulating Hormone (TSH), Luteinizing Hormone (LH), Testosterone, Follicle Stimulating Hormone (FSH), Beta-HCG, Sex hormone-binding globulin (SBHG), Rubella G, Alpha Fetal Protein (AFP), Prolactin, and many more. However, for this posting, I won't go into each test individually. I will mainly focus on the principles of the analyzer, only. Elecsys 2010 works based on 3 types of principles; competitive binding, sandwich binding and bridging binding.
Elecsys 2010 by Roche Diagnostics, USA.
Retrived 24th October 2008 from http://images.google.com.sg/imgres?imgurl=http://www.rochediagnostics.pl/content/produkty_i_uslugi/diag_scentr/immunodiagnostyka/img/elecsys_2010.jpg&imgrefurl=http://www.rochediagnostics.pl/content/produkty_i_uslugi/diag_scentr/immunodiagnostyka/elecsys2010.html&h=170&w=232&sz=17&hl=en&start=7&usg=__VI1tMedzGExoyIfrzsys6Kpc8Dg=&tbnid=BQz3cQjiytYxSM:&tbnh=80&tbnw=109&prev=/images%3Fq%3Delecsys%2B2010%2Broche%26gbv%3D2%26hl%3Den
- the analyzer uses this principle for analytes with low molecular weight (eg. Free T3)
Firstly, the antigen found in the sample (human serum) is mixed with specific anti-T3 antibody labelled with ruthenium complex. After incubation, the 2 particles will bind to one another. Another reagent is added, this time containing biotinylated T3 particles and streptavidin-coated paramagnetic microparticles. The process goes through another incubation cycle, after which immune complexes are formed. The immune complexes are transported into a measuring cell where they are magnetically entrapped onto a working electrode. The unbound particles are washed away by a washing reagent called the ProCell. The ruthenium complexes are then electrically stimulated to cause a chemiluminescent reaction to produce a light signal. The analyzer then detects the amount of light produced and calculates the concentration of antigens present in the human serum. The amount of light produced is indirently proportional to the amount of antigen present. The following is an illustration done using microsoft powerpoint of how the antibodies and antigens compete with one another for binding sites.2) Sandwich principle
- the analyzer uses this principle for analytes with higher molecular weight (eg. TSH)
The sample (human serum) containing antigens is mixed with biotinylated TSH antibodies and ruthenium-labeled TSH-specific antibodies. The mixture is then incubated for 9 minutes to allow the particles to bind to one another, after which another reagent containing streptavidin-coated paramagnetic microparticles are added and bind to the binding site of the biotinylated antibodies. The reaction goes through another incubation cycle causing the formation of immune complexes. The immune complexes are then transported into a measuring cell where they are magnetically entrapped onto a working electrode. The unbound particles are washed away by a washing reagent called the ProCell. The ruthenium complexes are then electrically stimulated to cause a chemiluminescent reaction to produce a light signal. The analyzer then detects the amount of light produced and calculates the concentration of antigens present in the human serum. The amount of light produced is dirently proportional to the amount of antigen present. The following is an illustration done using microsoft powerpoint of how the sandwich formation is formed between the antigens and antibodies.
3) Bridging principle
- the analyzer uses this principle when the tests require detection of antibodies and not antigens in the serum.
Firstly, the machine will detect the presence of serum antibodies in the sample. The serum antibodies are then mixed with biotinylated antigen found in the test reagent and ruthenium-labeled antigens. After a period of incubation to allow the particles to bind to specific binding sites, stretavidin-coated paramagnetic particles are added and bind to the biotinylated antigen binding site, forming immune complexes. The immune complexes with the paramagnetic particles bound to them will be magnetically attracted to a working electrode when they are transported into a measuring cell. Unbound complexes are washed away by ProCell. The ruthenium antigens are then electrically stimulated to produce light signals. The light signals are detected by the analyzer which will then calculate the antibodies concentrations in the human serum. The amount of light detected is directly proportional to the amount of antibodies present. The following is an illustration done using microsoft powerpoint, of how the bindings occur during the process. I guess that's all for you. Hope to see all of you soon.
2 more weeks of SIP C:C:C:C:C:C:
All cells undergo the cell cycle, a process by which the cell replicates through different phases. One of which is the M phase, where an adult cell splits into two daughter cells. The M phase is further categorized into two different processes: mitosis, where cell's chromosomes are divided between the two daughter cells, and cytokinesis, where the cell's cytoplasm divides and forms distinct cells. Prometaphase, metaphase, anaphase and telophase are stages that take place within mitosis.
M Phase in Cell Cycle
Metaphase is a stage in which condensed chromosomes align in the middle of the cell before being separated into each of the two daughter cells. Because the structure of chromosomes is the clearest at this stage, cells will be stimulated to stop growing at this stage for analysis. The metaphase chromosomes can be studied in spontaneously dividing cells or in cells that have been stimulated to divide in culture.
3 general processes that must take place in order to study the chromosomes are:
1) Culturing – Supplying the cells with nutrients to allow to proliferation
Before obtaining the metaphases of cells, cultures may be needed. The decision of whether to culture the cells lies on the specimen type. Spontaneously dividing samples such as bone marrow and lymph node may be set up for a direct harvest. Other samples such as tissues may require culturing for several days to allow the cells to proliferate. Once the sample has been cultured, harvesting is performed.
2) Harvesting – Obtain metaphases of cells, the stage at which chromosomes can clearly be seen
When cells are growing spontaneously, they will be harvested to obtain metaphases for analysis. In harvesting, three standard protocols will be used: mitotic arrest with Colcemid, hypotonic treatment with KCl (potassium chloride) and fixation with 3:1 methanol: acetic acid.
a) Mitotic arrest with Colcemid – Cells are arrested at the metaphase stage to enable the capturing and analysis of the chromosomes. Colcemid is used to prevent spindle formation, a process by which sister chromatids are pulled to opposite poles for incorporation of into the 2 daughter cells. It also promotes chromosome condensation, a process that can be affected by increased exposure time and concentration.
Different exposure time to Colcemid can affect the quality and quantity of the chromosomes; the condensation effect is greater when exposed for a longer period of time (ie. chromosomes are smaller in size but more). However, different culture and harvest methods may react differently to the Colcemid reaction. Therefore, to obtain the desired result, cells will be exposed to Colcemid for:
2 hours (long exposure time: more but shorter chromosomes)
20 mins (short exposure time: lesser but longer chromosomes)
b) Hypotonic Treatment with KCl – After arresting the cells at metaphase stage, they need to be treated with a hypotonic saline solution to increase the cell volume so that chromosomes can spread out. However, prolonged exposure may cause weakening of the cytoplasmic membrane, thus increasing the risk of the cell bursting and chromosomes to escape.
c) Fixation (3:1 methanol: acetic acid) – The purpose of fixing the cells is to remove the water content as well as to preserve them be hardening the membranes and chromatin, and somehow prepares chromosomes for the bending procedure.
3) Karyotyping – Chromosomes will be pair up to check for abnormalities
After fixing the cells, the slide needs to be stained to be able to visualize the bands on the chromosomes. Stained slides can now be karyotyped.
Down Syndrome (extra chromosome 21)
Klinefelter Syndrome (extra sex chromosome X)