Week 19 posting

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
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
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
-ABI Dye
-Sequencing Buffer
-Primer (Forward/Reverse)
-DNA/PCR product
-Distilled Water

Ethanol Precipitation
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
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.

Genotyping
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
Heterozygous: AG
Homozygous variant: GG

Liyanah Zaffre
0607718D
TG02