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BIOL1133 Biological Sciences Laboratory Course Practical 1 DNA sequencing and analysis Date of practical: 24-1-2013 Plasmid DNA no.: 16 Abstract Automated sequencing with dye-terminator was used in this practical.

Different length of DNA fragments was synthesized with fluorescently labeled ddNTP complementary to the template DNA. The DNA fragments were then separated by capillary electrophoresis and computer found out the sequence. The sequence was then matched with GenBank using BLAST. Finally the sample DNA sequence was found to be Mus musculus zinc finger protein (Sna) mRNA, complete cds.

Introduction DNA, i.e. deoxyribonucleic acid, is one of the most important molecules for life.

It controls the functioning as well as development of living things by carrying genetic information and expresses as protein to influence the biological status of the living organisms. Technique of DNA sequencing is important in acquiring the order of nitrogenous base in DNA. There are many DNA sequencing techniques nowadays, ranging from Chemical method (Maxam and Gilbert 1977) to Dideoxy method (Sanger). In this experiment, automated sequencing with dye-terminators was used to acquire the DNA sequence of an unknown gene which had been previously cloned into the given plasmid DNA, and the gene was identified accordingly.

The experiment was divided into two main parts. The first part was to perform automated sequencing with dye-terminators, which makes use of the special helical structure of DNA. Nucleotides are the basic component of DNA, consisting of three components, a 5-C deoxyribose, a nitrogenous base and a phosphate group attached to the 5’-end. The nucleotides are linked together by the phosphate group and the hydroxyl group at the 3’-end of the next nucleotide (Saenger & Wolfram 1984).

Several components required for the automated sequencing method are included in the substrates of the dye terminator, namely the given plasmid DNA, template DNA, primer, DNA polymerase, free normal dNTPs and small amount of fluorescently-labeled ddNTPs. ddNTPs, i.e. dideoxy nucleotides, are nucleotides

without 2’ and 3’ hydroxyl group to prevent continuous elongating once added to the end of a DNA strand. Such dideoxy sequencing is based on the primer-initiated synthesis of DNA strands complementary to the template DNA of unknown sequence (George et al. 1977). The primer would first attach to the template DNA, and then elongation will carry out under the presence of free nucleotides and the effect of polymerase. The elongation will stop at different lengths due to the presence of four types of dideoxy nucleotides, generating different sizes of DNA fragments. The elongation was performed under the following condition: Cycle sequencing: 1. 95 oC for 1 minute (denaturation of plasmid) 2. Repeat the following for 25 cycles: - 95 oC for 10 sec (denaturation of dsDNA) - 50 oC for 5 sec (annealing the primer and plasmid) - 60 oC for 2 min 30 sec (extension of the strand) 3. Hold at 4 oC The extension products were then purified using Ethanol/EDTA precipitation to remove the primers for the electrophoresis afterwards. The DNA fragments were separated by capillary electrophoresis by size (Swerdlow & Gesteland 1990), and then exposed to laser through detector cell. Different colours were generated due to the presence of four types of ddNTPs, and the computer will check for the pulses of the fluorescent colours. The collected data was used to generate a chromatogram to determine the DNA sequence of the given plasmid DNA. The second part of the experiment was the analyzing of the plasmid DNA sequence by a search tool BLAST. The plasmid DNA sequence was first read by the software Chromas Lite, and then the vector nucleotide sequence was removed to remain the gene of interest. Finally, the sequence of the unknown gene was allowed to match to the sequence in GenBand database with the use of BLAST via NCBI site.

Methods Please refer to the lab manual.

Experimental results and discussion

Chromatogram of plasmid DNA 16 (part) CTCCCTCACCCGCACCCTACTACTTCTGCCTACTGAGACCCACTGCTTACTGGCTTATCG AAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCATGCCGCGCTCCTTCCTG CTCCCTCACCCGCACCCTACTACTTCTGCCTACTGAGACCCACTGCTTAC 50 TGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTA 100 GCATGCCGCGCTCCTTCCTGGTCAGGAAGCCGTCCGACCCCCGCCGGAAG 150 CCCAACTATAGCGAGCTGCAGGACGCGTGTGTGGAGTTCACCTTCCAGCA 200 GCCCTACGACCAGGCCCACCTGCTGGCCGCCATCCCTCCGCCCGAGGTCC 250 TCAACCCCGCCGCTTCGCTGCCCACCCTCATCTGGGACTCTCTCCTGGTA 300 CCCCAAGTGCGGCCGGTTGCCTGGGCCACCCTCCCGCTGCGGGAGAGCCC 350 CAAGGCCGTAGAGCTGACCTCGCTGTCCGATGAGGACAGTGGCAAAAGCT 400 CCCAGCCGCCCAGCCCGCCCTCGCCGGCGCCGTCGTCCTTCACGTCCACC 450 TCGGCCTCCTCCCTGGAGGCCGAGGCCTTCATCGCCTTCCCTGGCTTGGG 500 CCAACTTCCCAAGCAGCTGGCCAGGCTCTCGGTGGCCAAGGACCCCCAGT 550 CGCGGAAGATCTTCAACTGCAAATATTGTAACAAGGAGTACCTCAGCCTG 600 GGCGCTCTGAAGATGCACATCCGAAGCCACACGCTGCCTTGTGTCTGCAC 650 GACCTGTGGAAAGGCCTTCTCTAGGCCCTGGCTGCTTCAGGGCCACGTCC 700 GCACCCACACTGGTGAGAAGCCATTCTCCTGCTCCCACTGCAACCGTGCT 750 TTTGCTGACCGCTCCAACCTGCGTGCCCACCTCCAAACCCACTCGGATGT 800 GAAGAGATACCAGTGCCAGGGCCTGTGCCCGAACCTTCTCCCGCATGTCC 850 CTTGCTCCACAAGCACCAAGAGTCTGGCTGCTCCGGAGGCCCTTCGCTGA 900 CAAGCTTGCTACCGAGCCTCGGATCAACTTAGTCAAGTGATGTGCATTCT 950 GCAGATATCCAGCACAGTGCCGACGCTCGAGTTCTAGAAAGGGGGCCCCC 1000 GA 1002 Sequence of plasmid DNA 16

The gene map of Mus musculus zinc finger protein (Sna) mRNA, complete cds Accession

Description

Max

Total

Query E

Max

score score

cover

value

ident

M95604.1

Mus musculus zinc finger protein (Sna) mRNA, complete cds

1026

1026

99%

0.0

99%

CT010204.1

Mus musculus full open reading frame cDNA clone RZPDo836G0150D for gene Snai1, Snail homolog 1 (Drosophila); complete cds, incl. stopcodon

1024

1024

99%

0.0

99%

NM_011427.2 Mus musculus snail 1020 homolog 1 (Drosophila) (Snai1), mRNA >gb|BC034857.1| Mus musculus snail homolog 1 (Drosophila), mRNA (cDNA clone MGC:41141 IMAGE:5121591), complete cds

1020

99%

0.0

99%

X67253.1

1020

99%

0.0

99%

M.musculus sna mRNA

1020

BLAST results of the 4 transcripts with highest max score (NCBI 2013) The plasmid DNA being sequenced is plasmid DNA number 16. It has 902 bases from the sequencing results, excluding the nucleotide of the vector backbone. After comparing with the multiple cloning site of pcDNA3.1(+) vector, the nucleotide of the vector backbone has been excluded til the last RE site (Pme I). The bases from base number 660 onwards are also removed as the peaks in the chromatogram are relatively irregular. The remaining bases were analyzed by BLAST. From the BLAST result, the gene is believed to be Mus musculus zinc finger protein (Sna) mRNA, complete cds, which has 1541 bases.

Only the middle part of the sequence is copied to BLAST when finding matches of the sequence for higher accuracy in the result. It is because the bases at the beginning contains the sequence from the vector, and the bases at the end was not well resolved due to the limitation of the technology. 559 bases were used for BLAST and the gene found has initially 1541 bases. As a high percentage match 99% was resulted, it can already be sure that all 559 bases are from Mus musculus zinc finger protein (Sna) mRNA, complete cds.

It was assumed in the experiment that the primer sequence was specific only to the primer binding site but not to any part of the cloned gene. This can assure that the whole set of DNA fragments would be synthesized. It was also assumed that the synthesized DNA fragments were specific to the cloned gene. Moreover, it was assumed that there were no alternation or any mutation in the nucleotides during the whole process of the experiment, so the accuracy of the result was not affected.

Ethanol/EDTA precipitation method is used for purification of the extension products. EDTA is a chelating agent which binds with metal ions, which helps to cease

the sequencing process in the reaction mixture. Adding ethanol in high concentration makes the metal ions interact with phosphate group in the DNA more easily, as ethanol has a low dielectric constant. The negative charge of the PO3- was shielded and was easily removed from the solution by centrifugation.

Automated sequencing with dye-terminator was used in the practical because no radioactive substances need to be handled when compared to the original Sanger method. DNA fragments can be produced within a short period of time and so the result can be obtained in shorter time. It is more convenient and safe, as the sequencing is done automatically and the computer reads the result. Also, only one single reaction is needed in the dye-terminator method while the original Sanger method needs four reactions.

Conclusion A DNA strand can be sequenced using automated sequencing technique with dye-determinator. The gene inserted into the vector is most likely to be Mus musculus zinc finger protein (Sna) mRNA, complete cds, with 1541 bases.

References 1. Saenger, Wolfram (1984). Principles of Nucleic Acid Structure. New York: Springer-Verlag 2. George KS, Zhao X, Gallahan D, et al. 1997. Capillary electrophoresis methodology for identification of cancer related gene expression patterns of fluorescent differential display polymerase chain reaction 3. Swerdlow H, Gesteland R. 1990. Capillary gel electrophoresis for rapid, high resolution DNA sequencing. Nucleic Acids Res 18: 1415–9.

4. Shendure JA, Porreca GJ, Church GM. 2008. Overview of DNA sequencing strategies. Curr Protoc Mol Biol Chapter 7: Unit 7.1.