Nanoball squencing

Nanoball squencing

1. NANOBALL SQUENCING By SHRUSHTI JOSHI 174551

2. INTRODUCTION DNA nanoball sequencing is a high throughput sequencing technology that is used to determine the entire genomic sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs . Fluorescent probes bind to complementary DNA and the probes are then ligated to anchor sequences bound to known sequences on the DNA template.

3. PROCEDURE Isolation of the desired DNA and converting it into small segments having 400-500 base pairs ( bp ). Ligating the Adapter sequences to the DNA sequence and converting the sequence into circular fragments. Using the Rolling Circle replication , these circular fragments are allowed to replicate and produce single stranded circular fragments. The single stranded circular fragments thus produced are then allowed to concatenate head to tail order to produce a DNA Nanoball . The Nanoballs are then allowed to adsorb on to a Flow cell . Fluorescent probes are then allowed to ligate with the specific nucleotide sequence. The colour of the fluorescence at each location is then recorded using a high resolution camera. Using the Bioinformatics techniques the data is then analyzed. The genomic data is then assembled and the sequence is identified.

4. ADVANTAGES It uses High density array so that high concentration of DNA can be used. Sequencing reaction is not progressive so that the new probes can be added after removing the probe already given. Accurate amplification using High fidelity Phi 29 DNA polymerase .

5. DISADVANTAGES Short read length of the DNA sequences obtained ,short reads, especially for DNA high in DNA repeats, may map to two or more regions of the reference genome. Multiple rounds of PCR , can introduce PCR bias and possibly amplify contaminants in the template construction phase

6. RECENT STUDIES Lee et al. used this technology to find mutations that were present in a lung cancer and compared them to normal lung tissue. They were able to identify over 50,000 single nucleotide variants Roach et al. used DNA nanoball sequencing to sequence the genomes of a family of four relatives and were able to identify SNPs that may be responsible for a Mendelian disorder, and were able to estimate the inter-generation mutation rate. The Institute for Systems Biology has used this technology to sequence 615 complete human genome samples as part of a survey studying neurodegenerative diseases. National Cancer Institute is using DNA nanoball sequencing to sequence 50 tumours and matched normal tissues from pediatric cancers

7. REFERENCES Revolvy.com Dnamismatch.com Yourgenome.org Ncbi Wikipedia Research.net

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