4. Molecular Biology Techniques

What principles underlie PCR?

Nucleotide base pairing, 5’→3’ polymerase synthesis, DNA denaturation (~95°C), extension from 3’ OH, semi-conservative replication.

What are the 3 steps of PCR?

Denature, Anneal, Extend

What happens during denaturation and renaturation?

Heat separates DNA strands (breaks H-bonds); cooling allows complementary strands to reform.

What are the components of a PCR reaction?

Template DNA, forward & reverse primers, Taq polymerase, dNTPs, Mg²⁺ buffer.

What is the role of Taq polymerase?

Thermostable enzyme that synthesizes DNA during high-temperature cycles (hot springs).

What is the role of primers in PCR?

Bind complementary sequences and define the target region for amplification.

What occurs in the first PCR cycle?

DNA strands separate, primers anneal, and DNA polymerase synthesizes new strands.

How does PCR amplification progress?

Exponentially (2ⁿ), producing millions to billions of DNA copies after many cycles.

What is the difference between forward and reverse PCR primers?

• Forward primer binds to the complementary strand and matches the 5’→3’ sequence of the coding strand (starts amplification).

• Reverse primer binds to the opposite strand and is reverse-complementary to the coding strand, allowing amplification in the opposite direction.

How are PCR products visualized?

Ethidium bromide intercalates DNA and fluoresces under UV light.

What is RT-PCR used for?

Amplifying RNA by converting it into cDNA using reverse transcriptase.

What is the process of RT-PCR?

RNA → cDNA → PCR amplification using Taq polymerase.

What is qPCR?

Quantitative (real-time) PCR that monitors DNA amplification cycle-by-cycle.

What does qPCR measure?

The amount of DNA (not just presence) using a fluorescent reporter probe.

What is Sanger sequencing?

A method of DNA sequencing using single-primer PCR and chain-terminating nucleotides.

What components are used in Sanger sequencing?

Template DNA, single primer, DNA polymerase, dNTPs, and ddNTPs.

What are dideoxynucleotides (ddNTPs)?

Chain-terminating nucleotides lacking a 3’ OH group.

Why do ddNTPs stop DNA synthesis?

“No 3’ OH = No extension”

What is the key mechanism in Sanger sequencing?

Competition between normal dNTPs and chain-terminating ddNTPs during DNA synthesis.

how are DNA fragments generated in Sanger sequencing?

Random incorporation of ddNTPs produces fragments of different lengths.

How are fragments analyzed in Sanger sequencing?

By capillary gel electrophoresis, separating DNA by size.

How is the DNA sequence determined?

By detecting labeled fragments (ddNTP or primer) and ordering them by size.

What are restriction enzymes?

Bacterial enzymes that cut DNA at specific sequences to protect against foreign DNA

What type of sequences do restriction enzymes recognize?

Palindromic sequences (4–8 base pairs long).

What bonds do restriction enzymes cut?

Phosphodiester bonds in both DNA strands.

What is a cloning vector?

What is the purpose of a cloning vector?

A DNA molecule that can be inserted into a cell and replicated.

To carry and replicate foreign DNA inside a host cell.

What are types of cloning vectors?

Plasmids, bacteriophages, cosmids, artificial chromosomes (bacterial, yeast, human).

What is rolling circle replication?

A mechanism by which plasmids replicate inside cells.

What are key elements of a vector?

Origin of replication, selectable marker, reporter gene, polylinker (MCS).

What is a selectable marker?

A gene that indicates if a vector was incorporated (e.g., antibiotic resistance).

What are common selectable markers?

Ampicillin (β-lactamase) and tetracycline (TetA efflux pump).

What is a reporter gene?

A gene used to determine if foreign DNA was inserted.

What is the lacZα system used for?

beta glactosidase encoded by lacza gene. Blue-white screening to detect successful insertion of DNA.

How does the blue-white test work?

X-gal is colorless; intact lacZ → blue product, disrupted lacZ → white colonies, DNA.

What is a polylinker (multiple cloning site, MCS)?

A short DNA sequence in a vector containing multiple restriction enzyme sites that allows insertion of foreign DNA.

What is Southern blotting used for?

Detecting specific DNA sequences and analyzing their size and quantity.

How does Southern blotting work?

DNA is digested, separated on a gel, transferred to a membrane, and probed.

• DNA is cut with restriction enzymes, run on a gel, then denatured to single strands

• DNA is transferred to a membrane using capillary action (buffer moves upward through sponge → gel → membrane → paper towels)

• A labeled DNA probe hybridizes to the target sequence and is detected to determine size and amount of DNA

What is Northern blotting used for? hat can Northern blotting reveal?

Detecting RNA and analyzing gene expression.

Quantity and size of RNA and differences in tissue expression.

How is Northern blotting similar to Southern blotting?

Same steps, but uses RNA instead of DNA.

What is Western blotting used for?

start eith tissue, virus or cell isolation. Detecting specific proteins from cells, tissues, or viruses.

What are the steps of Western blotting?

• Proteins are isolated and separated by gel electrophoresis

• Proteins are transferred to a membrane

• A specific antibody binds the target protein, and detection shows its presence and size

What is Eastern blotting used for?

Detecting protein modifications such as glycosylation or phosphorylation.

How are proteins detected in Eastern blotting?

Using probes such as lectins (e.g., Concanavalin A) that bind modified proteins. follow with floureschently labeled probe of conA., pHA to detect mammalian glycoprotein.

How do blotting techniques differ?

Southern = DNA; Northern = RNA; Western = protein; Eastern = protein modifications.

What is FISH (fluorescence in situ hybridization)?

A technique using fluorescent probes to localize specific DNA sequences on chromosomes.

What is FISH used for?

Detecting mutations, chromosomal abnormalities, and gene locations.

What is karyotyping?

Visualization of chromosomes to determine number and structure.

What is Giemsa staining used for?

Producing banding patterns on chromosomes for analysis.

When are cells prepared for karyotyping?

After being arrested during cell division.

What can karyotyping detect?

Chromosomal aberrations such as abnormal number or structure.

What is isoelectric focusing? What is isoelectric focusing used for?

Separation of proteins in an acrylamide gel with a copolymerized pH gradient based on their isoelectric point (pI).

Studying proteins and separating them based on acidic and basic residue content.

How do proteins behave in isoelectric focusing?

They migrate until reaching their pI, where they have no net charge and stop moving.

What is the difference between light and dark banding in karyotyping (Giemsa staining)?

• Dark bands = AT-rich, gene-poor, more condensed (heterochromatin)

• Light bands = GC-rich, gene-rich, less condensed (euchromatin)

A proteinthat is in a ph region below its pI is positively chaged and will migrate towards the _____

cathode (-)