BIOPHYSICS QUESTION ANSWERS

DNA NANOBALL SEQUENCING

1. What is DNA nanoball sequencing?

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. The base order is determined via the fluorescence of the
ligated and bound probes.

2. What is the need of high-throughput Sequencing technologies?

These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply
than the previously used Sanger sequencing, and as such have revolutionised the study of
genomics and molecular biology.

3. What is the silicon wafer of flow cell coated with?

Silicon wafer is coated with silicon dioxide, titanium, hexamethyldisilazane (HMDS), and a
photoresist material.

4. Why DNA is fragmented?

Bioinformatics mapping of the sequencing reads is most efficient when the sample DNA
contains a narrow length range.
5. What is rolling circle replication and the enzyme used for it?

Rolling circle replication describes a process of unidirectional nucleic acid replication that can
rapidly synthesize multiple copies of circular molecules of DNA or RNA. the enzyme used is Phi
29 DNA polymerase.

6. How imaging is done DNA nanoball sequencing?

The fluorophore is excited with an arc lamp that radiates specific wavelengths of light towards
the flow cell. The wavelength of the fluorescence of each DNA nanoball is captured on a high
resolution CCD camera.

7. What are the advantages of DNA nanoball sequencing?

• 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.

8. What are the disadvantages of DNA nanoball sequencing?

• short read length of the DNA sequences are obtained with this method. Short reads,
especially for DNA high in DNA repeats, may map to two or more regions of the reference
genome.

• A second disadvantage of this method is that multiple rounds of PCR have to be used.
This can introduce PCR bias and possibly amplify contaminants in the template construction
phase.
9. Write any two applications of DNA nanoball sequencing?

• The Institute for Systems Biology has used this technology to sequence 615 complete
human genome samples as part of a survey studying neurodegenerative diseases

• the National Cancer Institute is using DNA nanoball sequencing to sequence 50 tumours
and matched normal tissues from pediatric cancers.

10. Write the significance of DNA nanoball sequencing?

Massively parallel next generation sequencing platforms like DNA nanoball sequencing may
contribute to the diagnosis and treatment of many genetic diseases. The cost of sequencing an
entire human genome has fallen with the DNA nanoball technology.

Sequencing the entire genomes of patients with heritable diseases or cancer, mutations
associated with these diseases have been identified, opening up strategies, such as targeted
therapeutics for at-risk people and for genetic counseling.
 CRISPR Questions:

Q.1) What is CRSIPR and what does it stands for?

ANS) C- CLUSTERED, R- REGULARLY , I- INTERSPACED ,S- SHORT, P- PALINDROMIC , R- REPEATS

Q.2) What is the action of CRISPR?

ANS) The CRISPR immune system works to protect bacteria from repeated viral attack via three
basic steps:

(1)ADAPTATION: when the virus invades the bacterial cell new spacer is derived from virus and
integrated into CRISPR sequence.

(2)PRODUCTION OF CRISPR RNA: when the gene is identified by the CRISPR system CRIAPR RNA
is formed

(3)TARGETING: CRISPR RNA guide molecular machinery to target and destroy viral genome.

Q.3) What are Spacer genes?

ANS) Spacers genes are fragments of DNA gathered from the viruses that had tried to attack the
cell.

Q.4) What is gRNA? Explain its role.

ANS) The guide RNA has RNA bases that are complementary to those of the target DNA
sequence in the genome. The Cas9 follows the guide RNA to the same location in the DNA
sequence and makes a cut across both strands of the DNA.

Q.5) What is the loci range of crispr?

ANS) CRISPR loci range is from 24 to 48 base pairs.

Q.6) What is cas9?

ANS) CAS 9 Is a complex which consist of cas genes and g. RNA which is activated against viral
genome.

Q.7) How does gene editing differ from gene silencing?

ANS) In gene silencing the viral gene is distrupted and destroyed. While in gene editing gene is
repaired and not destroyed.

Q.8) What diseases can be cured by crispr?

ANS) In Medicine:

CRISPR-Cas9 has a lot of potential as a tool for treating a range of medical conditions that have
a genetic component.

1. Cancer:

To make the T cells against the cancer, the researchers must snip out pieces of three genes
using CRISPR/Cas9 technology. The T cells are isolated from the patient's blood, the edit is
performed on the T cells, then the cells are injected back into the patient.

2. Eye disorder:

Leber congenital amaurosis is an eye disorder affecting the retina characterized by severe loss
of vision at birth. Gene Carrying viruses are injected directly into eyes, in order to delete the
gene ( RPE65 gene) that Is responsible for a form of lca.

In malaria:

CRISPR can be used to treat malaria. Researchers have created a mosquito that not only doesn’t
transmit malaria but also passes on this trait to 99.5 percent of its offspring. They used this
gene-editing technique- CRISPR to insert two genes into the insect’s genome to confer malarial
resistance.
Q.9) Why Crispr is preferred over other gene editing techniques?

ANS) Traditional gene targeting has been very valuable for studying genes and genetics,
however it takes a long time to create a mutation and is fairly expensive.

There are drawbacks to other techniques:

1) they require costly and time-consuming protein synthesis for each unique DNA target
sequence.

2) they cannot access certain DNA sites.

CRISPR is preferred over other gene editing techniques because it is the fastest, cheapest and
most reliable system for ‘editing’ genes.

Q.10) What are the ethical concerns related to Crispr?

ANS) Gene editing can correct the faulty dna before the child’s birth. Because crispr involves
making changes to the body’s genetic setup, it raises many unique ethical concerns. Ethical
implications arise when modifying the human genome. “One cannot make changes in human
genome”
 Two Photon Intravital Cell Imaging
Q1. Two photon microscopy was first described by whom and when was it made
commercially available?

Ans. Two photon microscopy was first described by Maria Goppert Mayer, and in 1996 it was
made commercially available by Bio Rad.

Q2. What are the major advantages of two photon microscopy over confocal microscopy?

Ans. They are; deeper penetration, lesser phototoxicity, and no out of focus light.

Q3. What is intravital microscopy?

Ans. Intravital Microscopy is a technique used to observe biological systems in vivo, i.e, the
tissues are not required to be removed from the body to be examined.

Q4. What is fluorophore?

Ans. It is a fluorescent chemical compound that re-emits light upon light excitation. A
fluorophore is used to stain the specimen to be observed in analytical methods like fluorescent
spectroscopy.

Q5. Which electromagnetic radiation is used to excite a fluorophore in two photon

microscopy?

Ans. Infrared radiation is used.

Q6. What is the depth penetration in a two photon microscopy that uses IR radiation?

And. The depth penetration in a two photon microscope that uses IR radiation is up to 1mm.
Q7. What is phototoxicity?

Ans. Phototoxicity is the damage that can be caused to tissues because of exposure to
electromagnetic radiation.

Q8. Name the main components of the two photon microscope?

Ans. They are;

• Single-box pre-chirped femtopulsed IR laser

• Beam translation optics

• Laser intensity controller

• Laser scanner

• Detectors

Q9. What are the biomedical applications of two photon microscope?

Ans. Two-photon excitation excels at imaging of living cells, especially within intact tissues such
as brain slices, embryos, whole organs, and even entire animals. Two-photon absorption is a
photophysical process with diverse applications in medicine

• Photodynamic therapy,

• Neurophysiology,

• Cell biology (microscopy, photo-activated drugs) and

• Biomedical engineering (fabrication of micro-needle arrays and tissue scaffolds).

Q10. What are the recent advancement in two photon microscopy?

Ans. TPM is a versatile tool in biology, researchers have developed probes for specific
applications. Two Photon Probes for lipid rafts, lysosomes, mitochondria, and pH, also have
been developed. A series of two-photon probes for the membrane, organelles, metal ions,
glucose, redox mediators, reactive oxygen species, and reactive nitrogen species derived from
appropriate fluorophores
 RNAi
1) What is RNAi?

RNAi is RNA interference. It is a biological process in which RNA molecules inhibit gene
expression, typically by causing the destruction of specific mRNA molecules. RNA interference
(RNAi) is a natural process that cells use to 'turn off' or silence unwanted or harmful genes.
Historically, it was known by other names, including co-suppression, post-transcriptional gene
silencing (PTGS), and quelling.

2) What are two types of RNA’s that are central to RNA interference?

There are two types of small RNA’s that are central to RNA interference. These are

• MicroRNA (miRNA)

A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22

nucleotides) found in plants, animals and some viruses that functions in RNA silencing and post-
transcriptional regulation of gene expression. A non-coding RNA (ncRNA) is an RNA molecule
that is not translated into a protein.

• Small interfering RNA (siRNA)

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a
class of double-stranded RNA molecules, 20-25 base pairs in length, similar to miRNA, and
operates within the RNA interference (RNAi) pathway. It interferes with the expression of
specific genes with complementary nucleotide sequences by degrading mRNA after
transcription resulting in no translation.

These small RNAs can bind to other specific messenger RNA (mRNA) molecules and either
increase or decrease their activity, for example by preventing an mRNA from producing a
protein.

3) What are the differences between microRNA and small interfering RNA?

Although both these RNA’s takes part in RNAi and interferes with the regulation of gene
expression, there are some differences between them. These are:
 microRNA Small interfering RNA (siRNA)

microRNA is single stranded it is double stranded

It is a non-coding strand It is a coding strand

It is endogenous (made inside the cells) Exogenous (taken up by cells, it enters via vectors
such as viruses)

miRNA can inhibit translation of many different mRNA sequences because its pairing is
imperfect.

siRNA typically binds perfectly to its mRNA target. It is a perfect match to the sequence.

4) What are some breakthroughs that led to discovery of RNAi?

The discovery of RNAi was preceded first by observations of transcriptional inhibition by

expressed in transgenic plants, and more directly by reports of unexpected outcomes in
experiments performed by plant scientists in the United States and the Netherlands in the early
1990s.In an attempt to alter flower colors in petunias, researchers introduced additional copies
of a gene encoding chalcone synthase, a key enzyme for flower pigmentation into petunia
plants of normally pink or violet flower color. The overexpressed gene was expected to result in
darker flowers, but instead produced less pigmented, fully or partially white flowers, indicating
that the activity of chalcone synthase had been substantially decreased. Further investigation of
the phenomenon in plants indicated that the downregulation was due to post-transcriptional
inhibition of gene expression via an increased rate of mRNA degradation. This phenomenon
was called co-suppression of gene expression, but the molecular mechanism remained
unknown.

After these initial observations in plants, laboratories searched for this phenomenon in other
organisms. Craig C. Mello and Andrew Fire reported a potent gene silencing effect after
injecting double stranded RNA into C. elegans.] During the research, they found that injecting
mRNA that encodes for muscle protein production elicited no responses from the worms. Bear
in mind that the genetic code in the mRNA is considered as the sense sequence. They also tried
to inject antisense RNA into the worms which can pair with the sense sequence mRNA but it
also elicited no responses from the worms. Finally, when they tried to inject both the sense and
the antisense RNA together, they noted twitching movements from the worms. These results
surprised them since they know that the same kinds of movements were noted from worms
whose genes encoding for muscle protein were dysfunctional.
.In investigating the regulation of muscle protein production, they observed that neither mRNA
nor antisense RNA injections had an effect on protein production, but double-stranded RNA
successfully silenced the targeted gene. As a result of this work, they coined the term RNAi

5) How RNA interference play a role in gene knockdown?

The RNA interference pathway is often exploited in experimental biology to study the function
of genes in cell culture and in vivo in model organisms. Double-stranded RNA is synthesized
with a sequence complementary to a gene of interest and introduced into a cell or organism,
where it is recognized as exogenous genetic material and activates the RNAi pathway. Using
this mechanism, researchers can cause a drastic decrease in the expression of a targeted gene.
Studying the effects of this decrease can show the physiological role of the gene product.

6) How RNAi can be exploited in medicine?

It may be possible to exploit RNA interference in therapy. Although it is difficult to introduce

long dsRNA strands into mammalian cells due to the interferon response, the use of short
interfering RNA has been more successful. Among the first applications to reach clinical trials
were in the treatment of macular degeneration and respiratory syncytial virus.

Potential antiviral therapies include knockdown of host receptors and coreceptors for HIV, the
silencing of hepatitis A, and hepatitis B genes, silencing of influenza gene expression, and
inhibition of measles viral replication

Cancer: RNA interference is also a promising way to treat cancers by silencing genes
differentially upregulated in tumor cells or genes involved in cell division. A key area of research
in the use of RNAi for clinical applications is the development of a safe delivery method, which
to date has involved mainly viral vector systems similar to those suggested for gene therapy.

7) What I the role of RNAi in downregulation of genes.

Endogenously expressed miRNAs, are most important in translational repression and in the
regulation of

development,especially on the timing of morphogenesis and the maintenance of

undifferentiated or incompletely differentiated cell types such as stem cells.

In many organisms, including humans, miRNAs are linked to the formation of tumors and
dysregulation of the cell cycle. Here, miRNAs can function as both oncogenes and
tumorsuppressors
8) What are the active components of RISC?

The active components of an RNA-induced silencing complex (RISC) are endonucleases called
argonaute proteins, which cleave the target mRNA strand complementary to their bound
siRNA.

9) Is RNAi ATP dependent process? If yes explain.

The process proved to be ATP-independent and performed directly by the protein components
of RISC. However, an in vitro kinetic analysis of RNAi in the presence and absence of ATP
showed that ATP may be required to unwind and remove the cleaved mRNA strand from the
RISC complex after catalysis

10) Explain the mechanism of RNAi?

Endogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer, which binds and
cleaves double-stranded RNAs (dsRNAs) to produce double-stranded fragments of 20–25 base
pairs.These short double-stranded fragments are called small interfering RNAs (siRNAs). These
siRNAs are then separated into single strands and integrated into an active RISC complex. After
integration into the RISC, siRNAs base-pair to their target mRNA and cleave it, thereby
preventing it from being used as a translation template.
 Illumina DNA Sequencing
Q.1. Why Illumina DNA Sequencing is also known as Illumina Dye Sequencing?

It is known as illumine dye sequencing because this sequencing method is based on reversible
dye-terminators that enable the identification of single bases as they are introduced into DNA
strands.

Q.2. Why Illumina sequencing is considered as NGS technique?

NGS because this technique offers a number of advantages over traditional sequencing
methods such as Sanger sequencing. Due to the automated nature of Illumina dye sequencing it
is possible to sequence multiple strands at once and gain actual sequencing data quickly.

Q.3. Why adapters are added to the fragmented DNA Sample?

Adapters are added on either side of the cut points (ligation). Strands that fail to have adapters
ligated are washed away.[6]

Q.4. What is the importance of indices/terminal sequences in the Illumina sequencing?

Indices are usually six base pairs long and are used during DNA sequence analysis to identify
samples. Indices allow for up to 96 different samples to be run together. During analysis, the
computer will group all reads with the same index together.[7][8]

The terminal sequences are used for attaching the DNA strand to the flow cell. Illumina uses a
“sequence by synthesis” approach.

Q.5. What is the role of reversible terminators in the sequencing step of Illumina Sequencing?

A reversible terminator is on every nucleotide to prevent multiple additions in one round. Using
the four-colour chemistry, each of the four bases has a unique emission, and after each round,
the machine records which base was added.

Q.6. What are the uses of Illumina Sequencing?

It can also be used for whole-genome and region sequencing, transcriptome analysis,
metagenomics, small RNA discovery, methylation profiling, and genome-wide protein-nucleic
acid interaction analysis.[4
Q.7. What are the advantages and disadvantages of Illumina Sequencing?

ADVANTAGES

• Accurate

• Low error rate

• Low cost per base

• Tons of Data

Disadvantages:

• Large Scale

• Short Read Length (50-150bp)

• High Startup cost

Q.8. What is the importance of clustering in the Illumina Sequencing?

Clonal amplification is important for quality control purposes. If a strand is found to have an
odd sequence, then scientists can check the reverse strand to make sure that it has the
complement of the same oddity. The forward and reverse strands act as checks to guard
against artifacts.

Q.9. How are clusters generated in the Illumina Sequencing?

Clusters are generated through bridge amplification. Polymerases move along a strand of DNA,
creating its complementary strand. The original strand is washed away, leaving only the reverse
strand. At the top of the reverse strand there is an adapter sequence. The DNA strand bends
and attaches to the oligo that is complementary to the top adapter sequence. Polymerases
attach to the reverse strand, and its complementary strand (which is identical to the original) is
made. The now double stranded DNA is denatured so that each strand can separately attach to
an oligonucleotide sequence anchored to the flow cell. One will be the reverse strand; the
other, the forward. This process is called bridge amplification, and it happens for thousands of
clusters all over the flow cell at once.

Q.10. Why Illumina Sequencing is inexpensive then other sequencing techniques?

Additionally, this method only uses DNA polymerase as opposed to multiple, expensive
enzymes required by other sequencing techniques (i.e. pyrosequencing).