molecular bio pratice exam

b. reverse transcription

• This occurs in retroviruses (like HIV) and is mediated by the enzyme reverse transcriptase.

1. Which of the following is the process by which a single-stranded DNA is synthesized using RNA as
the template?
a. Transcription
b. Reverse transcription
c. Replication
d. Transformation
e. None of these is correct.

d. B and C

2. An encounter complex describes
a. Two atoms involved in a covalent bond.
b. An aspect of enzyme substrate binding.
c. A stage in the formation of a protein dimer.
d. B and C

reverse transciption

the process in which an RNA template is used to synthesize DNA.

encounter complex

an intermediate stage where molecules (such as an enzyme and its substrate, or two proteins) come together before forming a stable interaction

transcription

the process of making RNA from DNA, not the other way around.

replication

the process of making a DNA copy from DNA, not from RNA.

transformation

refers to the uptake of foreign DNA by a bacterial cell.

enzyme-substrate binding

involves an initial weak interaction before the substrate fully binds.

protein dimer formation

involves an intermediate stage before the two proteins fully associate.

c. reduced

• Recombination frequency depends on gene distance:

  • If two genes are far apart, recombination is more likely.

  • If two genes are close together, recombination is less likely.

• If genes are right next to each other, crossing over between them is very rare.

3. With a decreasing distance between two genes on a chromosome, the probability of a
recombination event is:
a. increased.
b. guaranteed.
c. reduced.
d. no more likely or less likely.
e. both increased and guaranteed.

e. as an indicator that the plasmid is recombinant, containing a DNA insert

• lacZ encodes β-galactosidase, which breaks down X-gal, producing a blue color.

• If a foreign gene is inserted, it disrupts lacZ, resulting in white colonies instead of blue.

• (c) Cutting the plasmid at one site depends on restriction enzyme sites, not lacZ.

In a plasmid vector, the purpose of the lacZ gene is:
a. to allow replication of the plasmid.
b. to allow selection of transformed bacterial cells.
c. to allow the plasmid to be cut once for cloning a fragment.
d. to protect the plasmid from digestion by exonucleases.
e. as an indicator that the plasmid is recombinant, containing a DNA insert.

replication of the plasmid

controlled by the origin of replication.

selection of transformed cells

done using antibiotic resistance genes.

b. tetS and ampR

• The plasmid contains tetR (tetracycline resistance) and ampR (ampicillin resistance).

• The EcoRI cut is inside the tetR gene, meaning that inserting Xenopus DNA disrupts tetR.

• This results in:

  • Loss of tetracycline resistance (tetS)

  • Ampicillin resistance remains (ampR)

Suppose that a plasmid which carries two genes for antibiotic resistance, tetR and ampR, is digested at its sole EcoRI site located within the tetR gene. DNA from the toad Xenopus is cut with EcoRI and the fragments allowed to ligate with the digested vector DNA. Bacteria are then transformed and grown on media lacking either antibiotic. What is / are the possible antibiotic resistance phenotype(s) of the bacteria in the culture?
a. tetR and ampR
b. tetS and ampR
c. tetS and ampS
d. All the above

b. gene encoding DNA polymerase

• A good plasmid vector should have:

  • (a) Multiple cloning site (MCS) → Provides multiple enzyme cut sites.

  • (c) Antibiotic resistance gene → Helps in selecting transformed bacteria.

  • (d) Origin of replication (ori) → Ensures plasmid replication.

  • (e) Blue/white selection system → Helps identify recombinant plasmids.

• DNA polymerase genes are not needed, as the host cell provides polymerase.

Which of the following is NOT a good feature of a plasmid vector?
a. Multiple cloning site
b. Gene encoding DNA polymerase

c. Antibiotic resistance gene
d. Origin of replication
e. Blue/white selection system

e. both the temperature at which the reaction occurs and the ionic strength of the hybridization buffer

Whether the probe is DNA or RNA does not directly affect stringency.

The stringency of a nucleic acid hybridization reaction is determined by:
a. whether the probe is DNA or RNA.
b. the temperature at which the reaction occurs.
c. the ionic strength of the hybridization buffer.
d. the lengths of the probe and the target sequence.
e. both the temperature at which the reaction occurs and the ionic strength of the hybridization buffer.

stringency

refers to how strictly a probe binds to its complementary DNA/RNA:

• High temperature = High _______ (only perfect matches bind).

• Low ionic strength = High ________ (reduces mismatched binding)

b. dideoxynucleotides

• Sanger sequencing (chain termination method) uses:

  • Dideoxynucleotides (ddNTPs) → Stop DNA synthesis at specific bases.

• PCR lacks ddNTPs because it aims to amplify the entire gene, not terminate synthesis at random points.

Which of the following molecular components would be present in a DNA sequencing reaction by the chain termination method, but NOT in a PCR reaction to amplify a particular gene?
a. DNA polymerase
b. Dideoxynucleotides
c. Template DNA
d. Deoxynucleotides
e. At least one DNA primer

e. C and D

• RNA in a non-denaturing gel folds into secondary structures, affecting mobility.

• Denaturing gels (e.g., formaldehyde gels) remove structure, so RNA is separated only by size.

• Charge (A) and Base composition (B) do not determine RNA migration in non-denaturing gels.

In non-denaturing gel electrophoresis, RNA molecules are separated by
A. charge
B. base composition
C. size.
D. secondary structure
E. C and D

b. southern blotting

The technique of _______________________ is used to transfer DNA from an agarose gel to a membrane.
a. RT-PCR
b. Southern blotting
c. restriction mapping
d. western blotting
e. northern blotting

southern blotting

detects specific DNA sequences

RT-PCR

amplifies RNA

western blotting

detects proteins

northern blotting

detects RNA

b. 5' GCAACG 3'

Which of the following sequences is inversely palindromic?
a. 5' GCATGC 3'
b. 5' GCAACG 3'
c. 5' GCAT 3'
d. 5' GCAACGC 3'
e. 5' GGAAAAGG 3'

inverted palindrome

read the same on both strands in opposite directions

e. none of the above could be inserted

A restriction enzyme cuts the following sequence 5'G/CATGC in a vector and leaves 4 bp overhangs. If kangaroo DNA is cut with five restriction enzymes each recognizing one of the shown sequences and leaving 4 bp overhangs, which ensuing fragments could NOT be cloned into the vector? For each sequence only one strand is shown and the slash indicates the location
of the cut site. (Hint: draw out at least the cut vector sequence as double-stranded DNA)
A. 5'T/CATGA
B. 5'C/CATGG
C. 5'A/CATGT
D. 5'C/GTACG
E. None of the above could be inserted.

c. map DNA sequences and genes to a chromosomal position

• FISH uses fluorescent probes to locate specific DNA sequences on chromosomes.

• It is used in genetic mapping and disease diagnosis.

Fluorescent in-situ hybridisation (FISH) is used to:
a) Map the location of chromosomes undergoing metaphase
b) Map DNA sequences from a specific parent in an individual
c) Map DNA sequences and genes to a chromosomal position
d) Map transcription factor binding sites on a chromosome
e) Map actively transcribed genes in heterochromatin

A molecular probe is a short, single-stranded DNA or RNA molecule that is designed to identify and hybridize (bind) specifically to a complementary sequence of nucleic acids. They are used in molecular biology and genetics for gene expression analysis, detecting genetic mutations, and studying the structure and function of nucleic acids

Describe a (molecular) probe and explain its use to identify a specific nucleic acid sequence.

In situ hybridization is a technique to study gene expression, identify specific RNA or DNA sequences, detect RNA or DNA molecules within cells or tissues. Various procedures are involved in preparing samples, as well as in detecting and analyzing them, which include:
1. Fixation of sample: to preserve the samples morphology and prevent degradation of nucleic acids, the samples are typically embedded in a suitable matrix (paraffin or cryosectioning compound) and sectioned thinly using a microtome to obtain tissue sections suitable for analysis.
2. Probe design and labeling: a complementary nucleic acid probe is designed for the target sequence of interest. The probe can be DNA or RNA and is usually labeled with fluorescent dyes, radioactive isotopes, or enzymes.
3. Hybridization: The labeled probe is applied onto the tissue sections or fixed cells and subjected to allows the probe to bind specifically to the target RNA or DNA molecules. After hybridization, the samples are washed to remove any unbound or nonspecifically bound probes.
4. Detection: For detection appropriate methods is used based on the type of probe label. For example, fluorescently labeled probes can be visualized using fluorescence microscopy, radioactive probes can be detected using autoradiography, and enzymatically labeled probes can be detected using chromogenic or chemiluminescent substrates.

5. Analysis: The distribution and intensity of the signal in the tissue sections or cells are analyzed to idetect the localization and abundance (intensity of the signal) of the target nucleic acid sequences. This information can provide informationabout gene expression patterns, cellular localization of specific RNA, genomic organization, and molecular interactions within cells or tissues.

Describe the technique of in situ hybridization and its purpose.

A technique for determining the sequence of nucleotides in a DNA molecule. The Sanger sequencing method is based on DNA replication and termination using chain-terminating nucleotides. Different steps re involved in preparing samples, detecting and analyzing them, which include:
1. Template DNA Preparation: The DNA to be sequenced is denatured into single strands by heating. These single-stranded DNA molecules serve as templates for sequencing.
2. Primer Annealing: A short synthetic oligonucleotide primer, complementary to a region of the template DNA is annealed to the template strand.
3. DNA Synthesis: DNA synthesis is initiated by a DNA polymerase enzyme (a DNA polymerase I or a modified version of it) which extends the primer by adding complementary nucleotides one at a time along the template strand.
4. Incorporation of Chain-Terminating Nucleotides: Along with the four standard
deoxynucleotides (dATP, dTTP, dCTP, and dGTP) used in DNA replication, small amounts of chain-terminating dideoxynucleotides (ddATP, ddTTP, ddCTP, and ddGTP) (which are labeled with a fluorescent or radioactive tag) are added to the reaction mixture. These chain-terminating nucleotides lack the 3' hydroxyl group necessary for forming a phosphodiester bond with the next nucleotide, which halts DNA synthesis when they are incorporated into the growing DNA strand.
5. Gel Electrophoresis (polyacrylamide): The reaction products are then separated by gel electrophoresis based on their size.( smaller DNA fragments to migrate more quickly through the gel than larger ones).
6. Visualization and Analysis: after gel electrophoresis, the DNA fragments are visualized using appropriate methods depending on the label used for the dideoxynucleotides (e.g., autoradiography for radioactive labels or fluorescence detection for fluorescent labels). The DNA sequence is determined by reading the pattern of labeled fragments from the gel, with each band representing a terminated fragment corresponding to a specific position along the template
DNA strand.

Describe the principle behind the chain termination (Sanger) method of DNA sequencing.