Nanoball Sequencing is the advanced form of DNA sequencing used to determine the entire gene sequence of an organism. The DNA Nanoball sequencing technique uses the Rolling Circle model of bacterial replication to convert the DNA of genes into Nanoballs. Fluorescent probes are used to bind with the complementary sequences, then these markers are used to locate the sequence in the DNA template. DNA Nanoball technique allows sequencing large number of DNA segments very fast at low cost. DNA Nanoball sequencing is a very good technique for identifying Mutations in genes and Mendelian disorders.
The Protocol of Nanoball sequencing involves:
- 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.
Extraction of DNA
DNA from cells is extracted through cell lysis using tissue fragmentation. The DNA thus obtained is mega base pair long which is sonicated using Ultrasound to break it into segments at random intervals. The fragments are then separated using PolyAcrylamide Gel Electrophoresis (PAGE). After the PAGE separation, the separated fragments are purified by Gel extraction resulting in small length segments having 400 to 500 base pairs.
Creation of Circular DNA
After the DNA fragmentation, Adapter DNA sequences are allowed to attach with the DNA segment of unknown sequence. The DNA is then amplified using PCR. The DNA is then modified to create single stranded ends which can be joined together to form a circular shaped DNA. Restriction endonuclease is used to cleave the DNA strand at 13 bp site to form the linear segment. During the process 4 adapter molecules are allowed to bind with the DNA segment. The Circular DNA template now contains 4 adapters.
Rolling Circle Replication
The full circular DNA is then amplified into a long string of DNA through Rolling circle replication using the enzyme Phi29 DNA Polymerase. The newly synthesized DNA is then separated from the circular DNA template resulting in a long DNA with copies of the circular DNA.
Formation of Nanoball
The 4 adapter sequences are still in the DNA that contains Palindromic sequences. The adapter sequences hybridize so that the single strand to fold itself into a tight ball of DNA with 300 nanometers across. The Nanoballs remain separated each other preventing the tangling in the single stranded DNA. To get the DNA sequence, the DNA Nanoballs are attached to a Flow cell which is 25mm by 75 mm silicon wafer. The Silicon wafer is coated with Silicon dioxide, titanium, hexamethyldisilazane and a Photoresist material. The Nanoballs in the Flow cell are allowed to bind with Aminosilane to create high density DNA Nanoballs.
Imaging and Data analysis
The order of DNA sequence is determined after arrayed on the Flow cell. Oligonucleotide complementary to one of the adapter is added along with DNA ligase enzyme. The positions of different Nitrogen bases in the DNA probe is shown by the Fluorophore attached.
Adenine contains Red fluorophore
Cytosine with Yellow fluorophore
Guanine with Green fluorophore
Thymine with Blue fluorophore
Only the Probe with complementary nucleotide will binds with the corresponding nucleotide in the DNA. The Non binding probes will be washed away. By observing the Fluorescence, it is easy to locate the position and type of base in the DNA. After identification, the probe is removed from the DNA and another probe is added to locate other nucleotides.
The Flow cell is then imaged to identify the base attached to the DNA Nanoball. For this, an Arc lamp is used. It illuminates the Nanoballs with specific wavelength light. A high resolution CCD (Charge Coupled Device) camera collects the wavelength of florescence from the Nanoball. The image is then processed and the computer records the position of base depending on the colour of fluorescence.
The advantages of DNA Nanoball sequencing include:
- 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.