DNA Sequencing

Question 1

Describe the process of Sanger Sequencing

Answer 1

1. Used to sequence DNA fragments of 1000bp or less
2. DsDNA is denatured via heat and separated
3. A P32-labelled primer anneals to the template strand
4. DNA polymerase, 4 dNTPS and low concentration of one of 4 ddNTPS are added
5. 3 further reactions set up each containing a different ddNTP
6. Fragments of different sizes are synthesised in each reaction since ddNTP addition terminates synthesis due to a lacking 3’ -OH group.
7. Each reaction are put on a gel showing bands of different fragments.
8. No fragment will be of the same size in reactions with different ddNTPS.
9. Comparing all 4 gels together and starting from smallest fragment to largest, the sequence of nucleotides can be identified since the type of ddNTP is known.
10. The coding strand is complementary to the template sequence identified

Question 2

Main differences between Sanger and NGS illumina sequencing

Answer 2

1. Radioactivity not required since DdNTPS are fluorescently labelled a different colour for each base.
2. Liquid handling done by robots
3. DdNTP terminated fragments are separated through fine capillaries not gel.
4. A laser detects the colour of ddNTP fluorescent label on each fragment
5. A computer generates a colour generated chromatogram reading 5’-3’

Question 3

Goals of Sanger sequencing

Answer 3

1. To check recombinant plasmids after cloning reactions
2. To directly sequence PCR fragments prior to cloning
3. Genotyping for single nucleotide polymorphisms

Question 4

Explain the process of illumina NGS sequencing

Answer 4

1. The sequencing reaction occurs on a flow cell (size of a microscope slide)
2. DNA Oligos are fragments that are covalently bonded to the flow cell surface
3. DNA oligos are arranged in pairs
   Library preparation:
4. DNA wanted to be sequenced is broken into 100-200bp fragments with overlapping regions by sonication and short DNA adapters are added to the ends of each fragment by DNA ligase
5. Adaptors are complementary to the oligos on the flow cell and serve as priming sites for amplification
   Cluster generation:
6. The prepared sequencing library is loaded onto flow cell and only one type of adaptor binds to flow cell so all fragments are attached to the flow cell at the same end
7. DNApol and dNTPs are added and DNA synthesis generates a double stranded fragment. The template strand bound to the flow cell is washed away
8. The newly synthesised strand attaches its free end to another complementary oligo on the flow cell forming a bridge
9. Another round of DNA synthesis reforms the template and double stranded DNA in a bridge.
10. DNA is denatured resulting in two complementary single-stranded templates
11. Bridge amplification and synthesis is repeated to generate 1000s of templates
12. All the reverse strands are washed away leaving only identical forwards strands
    Sequencing by synthesis:
13. Sequencing primer is hybridised to the adapter sequence on each forward strand.
14. Differently coloured fluorescently labelled nucleotides are added to the flow cell one at a time
15. When each nucleotide binds its colour of fluorescence is detected and the cycle is repeated to determine the next nucleotide
    Data analysis:
16. Sequencing software generates sequencing reads and thanks to overlapping sequences the fragments can be pieced together to reconstruct the OG DNA sequence

Question 5

Explain what RNA-Seq is and how it is useful

Answer 5

1. RNA sequencing is the sequencing of RNA fragments which are often stored in cDNA libraries via reverse transcription
2. Useful in characterising gene expression patterns
3. Can detect alternative splicing