“Biotechnology and its Applications” is crucial for board and competitive exams because it links basic biological concepts (DNA manipulation, gene expression, and molecular tools) with real-world technologies like PCR, genetic engineering, CRISPR, and therapeutic applications. Most questions test both calculation/interpretation (e.g., PCR bands, fragment sizes) and conceptual understanding (e.g., HDR vs NHEJ, off-target control, insertional risks), making this chapter high-yield for CBSE and JEE/NEET.
15
Minutes
10
Questions
1 / -0
Marking
Q1. In an ideal PCR reaction the number of DNA copies after cycles follows . If the reaction begins with template molecules and runs for cycles with 100% efficiency, how many copies of the target DNA will be present?
(10,485,760)
(104,857,600)
(1,048,576)
(209,715,200)
Q2. A gene has exon 1 of length and exon 2 of length separated by an intron of . Primers are designed with the forward primer inside exon 1 and the reverse primer inside exon 2 so PCR amplifies across the exon–exon junction. After RT-PCR and agarose gel electrophoresis, which band pattern indicates contamination of the RNA sample with genomic DNA?
Single band at — indicates only cDNA (no genomic DNA contamination)
Single band at — indicates only genomic DNA template (no RT product)
No bands — indicates failed RT-PCR or degraded RNA
Two bands at and — indicates presence of both cDNA (spliced product) and contaminating genomic DNA
Q3. A circular plasmid of size was digested separately with EcoRI and BamHI. EcoRI digestion produced two fragments of and . BamHI digestion produced a single linear band of (one BamHI site). A double digest (EcoRI + BamHI) produced fragments of , and . Based on these data, where is the BamHI site located relative to the two EcoRI sites?
The BamHI site lies within the EcoRI fragment and divides it into and segments (i.e., BamHI is from one EcoRI site and from the other).
The BamHI site lies within the EcoRI fragment and divides it into and segments.
BamHI coincides with one of the EcoRI sites (double digest would then give and only).
The observed pattern (3,2,5 kb) is an artifact of partial EcoRI digestion; BamHI still cuts plasmid but EcoRI does not fully generate only the 3 kb and 7 kb fragments.
Q4. Statement I: "Integration of therapeutic genes into the host genome using integrating viral vectors (e.g., gammaretroviruses) can activate nearby oncogenes and has caused leukemia in some gene therapy trials." Statement II: "Long terminal repeats (LTRs) present in these integrating vectors reduce the risk of insertional activation of host oncogenes because they act as insulators that block enhancer activity."
Both statements are true and Statement II correctly explains Statement I.
Both statements are true but Statement II does not correctly explain Statement I.
Statement I is true but Statement II is false.
Statement I is false but Statement II is true.
Q5. For SpCas9 the guide RNA spacer is 20 nt; positions are numbered to with position adjacent to the PAM (PAM-proximal, seed region) and position PAM-distal. Two potential off-target genomic sites both have canonical NGG PAM. Off-target A has a single mismatch at position (near PAM), and off-target B has a single mismatch at position (PAM-distal). Which off-target is more likely to be cleaved by Cas9 and why?
Off-target A, because mismatches close to the PAM are more tolerated than those far from the PAM.
Off-target B, because mismatches in PAM-distal positions are tolerated more and the PAM-proximal (seed) region is most critical for cleavage.
Both are equally likely, because a single mismatch anywhere reduces cleavage probability to the same extent.
Neither is likely to be cleaved, because any mismatch between guide and target abolishes Cas9 activity.
Q6. In an ideal PCR each cycle doubles the number of DNA molecules. Starting with template molecules, how many molecules will be present after cycles? (Assume 100% efficiency and no limiting reagents; use .)
molecules
molecules
molecules
molecules
Q7. A circular plasmid of size has unique restriction sites at positions (in bp): EcoRI at and , and BamHI at . If the plasmid is digested simultaneously with EcoRI and BamHI, which set of fragment sizes (in kb) will be observed on an agarose gel?
and
single linear fragment of
three fragments of
three fragments of
Q8. Assertion (A): Delivering CRISPR–Cas9 as preassembled ribonucleoprotein (RNP) complexes into cultured cells reduces off-target mutations compared to delivering Cas9 via plasmid transfection.
Reason (R): RNP delivery produces a transient presence of active Cas9–guide complexes in the cell, whereas plasmid delivery leads to prolonged Cas9 expression and increases the time window for off-target cleavage.
Both A and R are true and R explains A
Both A and R are true but R does not explain A
A is true but R is false
A is false but R is true
Q9. You attempted CRISPR–Cas9 mediated knock‑in of a coding cassette into a mammalian locus. The donor template delivered was a single‑stranded oligonucleotide (ssODN) carrying the insert flanked by homology arms on each side. After screening many clones you observed numerous small indels at the target site but no precise knock‑ins. Which explanation best accounts for this outcome?
Cas9 failed to cut the target site, so HDR could not proceed
homology arms on an ssODN are sufficient for insertion of a fragment; failure indicates off‑target cleavage elsewhere
Inserting a large () fragment by HDR in mammalian cells typically requires a double‑stranded donor with much longer homology arms (hundreds of bp) and cells in S/G2; using a short ssODN favours NHEJ and produces indels instead
An ssODN donor cannot mediate HDR in mammalian cells under any circumstances; therefore only NHEJ repairs occur, producing indels
Q10. You must clone a coding sequence (CDS) into a plasmid whose multiple cloning site contains a unique EcoRI site. The CDS itself contains EcoRI sites at and . Which strategy will most reliably yield full‑length insert cloned in a defined orientation?
Digest both vector and CDS with EcoRI and ligate; screen colonies for full‑length inserts
PCR‑amplify the CDS using primers that add two different restriction sites (e.g., XhoI at 5' and HindIII at 3') that are absent in the CDS, digest PCR product and vector with those enzymes and perform directional ligation
Perform a partial EcoRI digest of the CDS to enrich for full‑length fragment and ligate into EcoRI‑digested vector
Use blunt‑end cloning (e.g., into an EcoRV site) without PCR so orientation is not controlled