17. Technique

Question 1

What is a restriction endonuclease and its natural function?

Answer 1

An enzyme that cuts DNA at specific sequences. Its natural function is part of the restriction-modification system in bacteria to degrade viral DNA.

Question 2

Explain the ‘restriction-modification system’ in bacteria.

Answer 2

A defense mechanism where restriction enzymes cut foreign DNA, while the bacterium’s own DNA is protected by methyltransferases that add methyl groups to the recognition sites.

Question 3

What is the role of DNA ligase in molecular cloning?

Answer 3

It acts as ‘molecular glue’ by catalyzing the formation of phosphodiester bonds to join DNA fragments (like an insert and a plasmid vector) together.

Question 4

Define a plasmid vector in the context of molecular cloning.

Answer 4

A small, circular, extrachromosomal DNA molecule used to carry foreign genetic material into a host cell for replication or expression.

A plasmid vector is a modified circular, double-stranded DNA molecule used as a tool in genetic engineering to isolate, multiply, or express specific DNA sequences within a host cell. Unlike chromosomal DNA, these molecules replicate independently within the cell. In the context of molecular cloning, plasmid vectors function as vehicles to carry foreign DNA fragments, which are joined to the vector using DNA ligase to create a recombinant molecule.

Essential features of a functional plasmid vector include:
* Multiple Cloning Site (MCS or polylinker): A short region containing several unique restriction sites that allow for the easy insertion of target DNA.
* Origin of Replication (ori): The specific site where DNA polymerase binds to initiate the autonomous replication of the plasmid.
* Selectable Markers: Genes that provide antibiotic resistance, allowing researchers to identify and select only the host cells that have successfully taken up the plasmid.

Once the target gene is inserted into the vector, the resulting recombinant plasmid is introduced into a host bacterium (such as E. coli) through transformation, where it is copied millions of times as the bacteria multiply.

Question 5

What is the role of X-Gal and IPTG in blue-white screening?

Answer 5

IPTG induces the expression of the lacZ gene. If the lacZ gene is functional, it produces beta-galactosidase which cleaves X-Gal, turning colonies blue. Recombinant colonies (white) indicate the gene was disrupted by an insert.

Question 6

Describe the three main steps of PCR (Polymerase Chain Reaction).

Answer 6

1. Denaturation: High heat separates DNA strands.(~95℃)
2. Annealing: Primers bind to target sequences.
3. Extension: Taq polymerase synthesizes new DNA strands.

Question 7

Why is Taq-Polymerase essential for PCR?

Answer 7

It is a heat-stable DNA polymerase (originally from Thermus aquaticus) that can withstand the high temperatures required for DNA denaturation without losing activity.

Question 8

What is DNA denaturation and how is it achieved?

Answer 8

The process of separating double-stranded DNA into single strands, typically achieved by heating the DNA to approximately 94-98 degrees Celsius.

Question 9

Explain the principle of Gel Electrophoresis.

Answer 9

A technique that separates DNA, RNA, or proteins based on size and charge by applying an electric field as they move through a porous gel matrix.

Question 10

Distinguish between Southern, Northern, and Western blotting.

Answer 10

Southern: Detects specific DNA sequences. Northern: Detects specific RNA sequences. Western: Detects specific proteins using antibodies.

Question 11

What is the specialized use of Eastern-blotting?

Answer 11

It is used to detect post-translational modifications of proteins, such as carbohydrate or lipid attachments.

Question 12

Compare Immunohistochemistry (IHC) and Immunocytochemistry (ICC).

Answer 12

IHC is used to visualize specific proteins in intact tissue sections, while ICC is used for isolated cells or cell cultures.

Question 13

What is an epitope?

Answer 13

The specific part of an antigen (usually a short sequence of amino acids) to which an antibody binds.

Question 14

Describe the difference between Immunofluorescence and Immunoenzymatic methods.

Answer 14

Immunofluorescence uses fluorescent dyes (fluorophores) as markers, while immunoenzymatic methods use enzymes (like peroxidase) to catalyze a color-producing reaction.

Question 15

What is Autoradiography?

Answer 15

A technique that uses X-ray film or digital sensors to detect radioactive substances within a biological sample (e.g., labeled DNA or proteins).

Question 16

Explain the ‘levels of hierarchy in biology’ in terms of techniques.

Answer 16

Refers to the transition from molecules (DNA/RNA/Protein) to organelles, cells, tissues, and organisms, requiring different analytical techniques at each level.

Question 17

What is a Genomic Library?

Answer 17

A collection of total genomic DNA from a single organism, stored in a population of identical vectors, each containing a different fragment of the DNA.

Question 18

Contrast a Genomic Library with a cDNA Library.

Answer 18

Genomic libraries: Collection of Clones, which contain all DNA (including introns and promoters).
→sequencing (genome projectino), isolation of genes
**cDNA libraries **are made from mRNA (a cDNA copy of each mRNA) and contain only the protein-coding sequences (exons).

Question 19

What are VNTR and STR analysis used for?

Answer 19

Variable Number Tandem Repeats and Short Tandem Repeats are used for DNA profiling, forensic identification, and paternity testing based on genetic variation.

Question 20

Describe the principle of DNA Sequencing (Sanger method).

Answer 20

Uses dideoxynucleotides (ddNTPs) to terminate DNA chain elongation at specific bases, allowing the determination of the exact sequence of nucleotides.

Question 21

What is the ‘Shotgun Technique’ in genomics?

Answer 21

A method for sequencing long DNA strands where the DNA is broken into random fragments, sequenced, and then reassembled using computer software.

Question 22

What is a DNA Microchip (Microarray) used for?

Answer 22

To simultaneously monitor the expression levels of thousands of genes or to detect specific genetic variations (SNPs) across a genome.

Question 23

Explain the advantage of Real-time PCR (qPCR) and RT-PCR.

Answer 23

Real-time PCR allows for the quantification of DNA in real-time. RT-PCR involves converting RNA to cDNA first, allowing for the quantification of gene expression (mRNA).

Question 24

What is the Klenow fragment?

Answer 24

A large fragment of E. coli DNA Polymerase I that retains polymerase and 3’ to 5’ exonuclease activity but lacks 5’ to 3’ exonuclease activity.

Question 25

Define the function of S1 Nuclease.

Answer 25

An enzyme that specifically degrades single-stranded DNA or RNA, often used to remove single-stranded 'tails' from DNA fragments.

Question 26

What is the role of Alkaline Phosphatase in cloning?

Answer 26

It removes 5' phosphate groups from DNA fragments to prevent self-ligation of linearized plasmid vectors.

Question 27

Explain Gel Retardation (EMSA).

Answer 27

Electrophoretic Mobility Shift Assay; used to detect DNA-protein or RNA-protein interactions by observing the slower migration of complexes compared to free nucleic acids.

Question 28

What is Footprint Analysis used for?

Answer 28

To identify the specific DNA sequence where a protein (e.g., a transcription factor) binds by protecting that region from enzymatic cleavage.

Question 29

Describe the ABC method in immunohistochemistry.

Answer 29

Avidin-Biotin Complex method; uses the high affinity of avidin for biotin to amplify the signal, increasing the sensitivity of protein detection.

Question 30

What is In Situ Hybridization (ISH)?

Answer 30

A technique using a labeled complementary DNA or RNA probe to localize a specific DNA or RNA sequence within a tissue or cell.

Question 31

What makes FISH (Fluorescence In Situ Hybridization) distinct?

Answer 31

It uses fluorescent probes to visualize and map genetic material, often used to detect chromosomal abnormalities in medical genetics.

Question 32

Explain FRET (Fluorescence Resonance Energy Transfer).

Answer 32

A technique to measure distances and interactions between two molecules by the transfer of energy from a donor fluorophore to an acceptor fluorophore.

Question 33

What is Flow Cytometry and its role in FACS?

Answer 33

Flow Cytometry measures physical and chemical characteristics of cells in a fluid stream. FACS (Fluorescence-Activated Cell Sorting) uses this data to physically sort cells into different groups.

Question 34

How is Green Fluorescent Protein (GFP) used as a tool?

Answer 34

Used as a biological marker to 'tag' proteins or organelles, allowing researchers to observe their location and movement in living cells in real-time.

Question 35

What is an EST Library?

Answer 35

Expressed Sequence Tag library; a collection of short sub-sequences of cDNA, used to identify gene transcripts and instrumental in gene discovery.

Question 36

Define RFLP (Restriction Fragment Length Polymorphism).

Answer 36

Variation in the length of DNA fragments produced by restriction enzymes, used as a genetic marker for mapping and disease diagnosis.

Question 37

What is ChIP (Chromatin Immunoprecipitation) used for?

Answer 37

To investigate interactions between proteins (like histones or transcription factors) and DNA in the natural context of the cell's chromatin.

Question 38

Explain DNA Methylation Analysis.

Answer 38

Techniques used to detect the addition of methyl groups to DNA (usually at CpG sites), which is a key epigenetic modification influencing gene expression.

Question 39

What is Methyl-Seq?

Answer 39

A high-throughput sequencing method specifically designed to map methylated cytosines across the entire genome.

Question 40

Multiple Choice: Which technique is used to detect RNA? A) Southern Blot B) Northern Blot C) Western Blot D) Eastern Blot E) PCR

Answer 40

Answer: B) Northern Blot

Question 41

Multiple Choice: Which enzyme is used to create cDNA from mRNA? A) Taq Polymerase B) DNA Ligase C) Reverse Transcriptase D) S1 Nuclease E) Alkaline Phosphatase

Answer 41

Answer: C) Reverse Transcriptase (Context of cDNA library)

Question 42

Multiple Choice: In PCR, what happens at the Annealing step? A) DNA strands separate B) DNA is synthesized C) Primers bind to DNA D) DNA is methylated E) Proteins are denatured

Answer 42

Answer: C) Primers bind to DNA

Question 43

Multiple Choice: Which method is best for sorting living cells based on size and fluorescence? A) IHC B) FRET C) FACS D) FISH E) Autoradiography

Answer 43

Answer: C) FACS

Question 44

Multiple Choice: GFP was originally isolated from which organism? A) E. coli B) Thermus aquaticus C) Aequorea victoria (Jellyfish) D) Yeast E) Fruit fly

Answer 44

Answer: C) Aequorea victoria (Jellyfish)

Question 45

Multiple Choice: What is the purpose of using ddNTPs in Sanger sequencing? A) To speed up the reaction B) To act as primers C) To terminate DNA chain growth D) To label the template E) To prevent denaturation

Answer 45

Answer: C) To terminate DNA chain growth

Question 46

Multiple Choice: Which technique detects protein-DNA interactions? A) ChIP B) Western Blot C) Northern Blot D) RFLP E) FACS

Answer 46

Answer: A) ChIP

Question 47

Multiple Choice: X-Gal is cleaved by which enzyme? A) DNA Polymerase B) Beta-galactosidase C) Restriction Enzyme D) Ligase E) Phosphatase

Answer 47

Answer: B) Beta-galactosidase

Question 48

Multiple Choice: Southern blotting requires which of the following? A) Antibodies B) DNA probes C) RNA probes D) Reverse transcriptase E) GFP

Answer 48

Answer: B) DNA probes

Question 49

Multiple Choice: STR analysis is primarily used in: A) Protein folding B) Forensic science C) Cell cycle analysis D) Microscopy E) RNA stability

Answer 49

Answer: B) Forensic science

Question 50

Multiple Choice: The 'S' in FISH stands for: A) Southern B) Sequence C) Situ D) Sorting E) Single

Answer 50

Answer: C) Situ

Question 51

What techniques detect protein-DNA interaction?

Answer 51

protein-DNA interactions: 1.** Chromatin Immunoprecipitation (ChIP)** ChIP is used to identify the specific DNA sequences that a particular protein, such as a transcription factor, is bound to within a cell . Mechanism: Cells are treated with formaldehyde to cross-link proteins to the DNA they are currently touching . The chromatin is then fragmented (sonicated), and an antibody specific to the protein of interest is used to precipitate the protein-DNA complex . Result: The DNA is purified and sequenced to determine the exact binding sites of the transcription factor across the genome . **2. Gel Retardation Analysis (EMSA)** Also known as a mobility shift assay, gel retardation is used to determine if a specific protein binds to a given DNA sequence . Mechanism: DNA samples are run on a gel in two versions: one with nuclear proteins added and one without . Result: If the protein binds to the DNA, it creates a larger complex that moves more slowly through the gel compared to the free DNA. This produces a "retarded band" higher up on the gel, indicating an interaction has occurred . **3. DNA Footprint Analysis** Footprint analysis is used not only to detect binding but to locate the exact binding site of a protein on a DNA molecule . Mechanism: DNA is radioactively labelled at one end. A protein is allowed to bind to the DNA, and then an enzyme called DNase I is added to cut the DNA at various points . Result: The protein bound to the DNA acts as a shield, protecting that specific segment from being cut . When the resulting fragments are separated by gel electrophoresis, the protected region appears as a gap or "footprint" in the otherwise regular pattern of DNA cleavage .