Comprehensive Cloning, PCR, and Genetic Engineering Techniques in Molecular Biology

What is recombinant DNA?

DNA made by combining fragments from different sources in vitro.

What is a vector in cloning?

A DNA carrier that replicates independently and delivers DNA of interest into a host cell.

Why are plasmids useful as vectors?

They replicate on their own, can carry inserts, and often include selectable markers.

What is ori?

Origin of replication — the site where DNA copying begins. Determines copy number and host range.

What is the difference between high-copy and low-copy origins?

Strong origins give high copy number (~100-200 copies); weak origins give low copy number (~2 copies).

What is ampR?

A resistance gene encoding beta-lactamase, which breaks down ampicillin. Used as a selectable marker.

What is a sticky end?

A staggered single-stranded tail left after restriction enzyme cutting. Matching sticky ends can base-pair.

What is a palindromic site?

A DNA sequence read the same in opposite directions on complementary strands. Restriction enzymes recognize these.

What does DNA ligase do in cloning?

Seals the sugar-phosphate backbone to join DNA fragments into recombinant DNA.

Why use cDNA instead of genomic DNA for many cloning projects?

cDNA reflects the coding sequence without introns, making it easier to study protein-coding genes.

What enzyme makes cDNA from mRNA?

Reverse transcriptase.

How is the second strand of cDNA completed?

RNase H partially degrades the mRNA template; then DNA polymerase and DNA ligase complete the second strand.

What is a DNA library?

A collection of recombinant vectors containing DNA inserts — either genomic or cDNA.

What is the difference between a genomic library and a cDNA library?

A genomic library starts with chromosomal DNA; a cDNA library starts with cDNA made from mRNA.

What is PCR?

Polymerase chain reaction — a method that makes many copies of a specific DNA segment in vitro.

What are the three PCR steps in order?

Denaturation (heat separates strands), Annealing (primers bind), Extension (polymerase builds new strand).

What does Taq polymerase do in PCR?

Extends new DNA strands at high temperatures because it is heat-stable.

How many copies does PCR produce after n cycles?

Approximately 2^n copies.

What is RT-PCR used for?

Amplifying and detecting RNA by first converting it to cDNA with reverse transcriptase.

What does real-time (qPCR) measure?

The amount of DNA or mRNA in a sample, quantified by fluorescence during each cycle.

How do TaqMan probes work in qPCR?

A reporter and quencher are on the same probe; Taq cleaves the quencher during extension, allowing the reporter to fluoresce.

What does a low Ct value mean in qPCR?

A high starting amount of template DNA (fewer cycles needed to reach the threshold).

What does a high Ct value mean in qPCR?

A low starting amount of template DNA (more cycles needed).

What is a dideoxynucleotide and what does it do?

A chain-terminating nucleotide — when incorporated into a growing strand, elongation stops.

What is site-directed mutagenesis?

A technique to introduce a specific mutation at a chosen site, used to study gene function.

What are the steps of site-directed mutagenesis?

Make DNA single-stranded → use a mismatched primer → amplify by PCR → transform host → identify clones by sequencing.

What is CRISPR-Cas?

A bacterial immune mechanism adapted for targeted genome editing using guide RNA and the Cas9 nuclease.

What does Cas9 do?

Makes a double-strand break at the target DNA sequence directed by a guide RNA (sgRNA).

What is sgRNA?

Single guide RNA — a combined tracrRNA + crRNA that directs Cas9 to its target.

What are the two DNA repair outcomes after a Cas9 cut?

HRR (homology-directed repair) for precise edits, or NHEJ (non-homologous end joining) for disruption/knockout.

What does Northern blotting detect?

RNA — it separates RNA by size and detects a specific transcript using a labeled DNA probe.

What does Western blotting detect?

Protein — it separates proteins by size and detects a specific protein using antibodies.

What does EMSA test?

Electrophoretic mobility shift assay — tests whether a protein binds RNA or DNA; bound DNA migrates more slowly.

What does DNase I footprinting reveal?

The exact DNA region protected by a bound protein (protein blocks DNase I digestion, leaving a "footprint").

What is biotechnology?

The use of living organisms or their products to make useful products or carry out useful processes.

What is a transgenic organism?

An organism that has had a gene from another species transferred into it for a specific purpose.

What are recombinant organisms used for?

Improving strains, making novel products, medicine, biological control, and pollution reduction.

What is a classic medicine example from biotechnology?

Genetically engineered human insulin produced by Genentech.

What is Bt (Bacillus thuringiensis) used for?

Biological control — it produces toxins that kill caterpillars and beetles but are harmless to plants and animals.

What is environmental remediation in biotech?

Using microbes to extract heavy metals (Cu, Pb, Ni) or break down toxic chemicals like chlorinated hydrocarbons.

What is gene knockin?

Inserting a gene into a noncritical chromosomal site.

What is gene knockout?

Inactivating a copy of a gene to study its function.

What is xenotransplantation?

Transplanting tissues, cells, or organs from one species into another.

What is molecular pharming?

Engineering mammals to produce medically important proteins in their milk glands.

What is reproductive cloning?

Creating two or more genetically identical individuals (e.g., embryo splitting or somatic cell nuclear transfer).

What are stem cells and why are they medically valuable?

Undifferentiated cells that can divide and become specialized — valuable for regenerative medicine and tissue repair.

How are many transgenic plants made?

Using Agrobacterium tumefaciens, which naturally inserts a plasmid into plant cells (tumor gene replaced with recombinant DNA).

What is gene therapy?

Introducing cloned genes into a patient to compensate for mutant or missing genes.

What is the advantage and disadvantage of viral gene therapy?

Advantage: high efficiency. Disadvantage: risk of triggering an immune response.

What is the advantage and disadvantage of liposome (nonviral) gene therapy?

Advantage: no immune response. Disadvantage: lower efficiency.

What does ADA gene therapy involve?

Removing ADA-deficient lymphocytes → culturing them → infecting with retrovirus carrying normal ADA gene → returning corrected cells to patient.

What is genomics?

The molecular analysis of the entire genome of a species, including nuclear and extranuclear genetic material.

What are the three genome mapping strategies?

Cytogenetic mapping, linkage mapping, and physical mapping.

What is cytogenetic mapping?

Using microscopy (Giemsa banding or FISH) to locate sequences on chromosomes.

What is FISH?

Fluorescent in situ hybridization — uses fluorescent probes to locate specific sequences on chromosomes.

What is linkage mapping?

Using recombination frequency from genetic crosses to determine relative gene positions.

What is the formula for map distance?

(Recombinant offspring ÷ Total offspring) × 100 = map units (centiMorgans).

What is physical mapping?

Using cloned fragments to determine actual distances and locations by finding overlapping regions.

What is a contig?

A set of overlapping cloned DNA pieces that together represent a continuous chromosomal region.

What is a YAC?

Yeast artificial chromosome — a large-insert vector holding several thousand to 2 million bp.

What is a BAC?

Bacterial artificial chromosome — a large-insert vector holding ~300,000 bp inserts.

What is shotgun sequencing?

Breaking DNA into random fragments, sequencing each, then assembling the sequence by finding overlaps.

What is pyrosequencing?

A next-generation sequencing method based on detection of pyrophosphate released during nucleotide incorporation.

What is metagenomics?

Studying genetic material collected directly from environmental samples containing many organisms.

Why is metagenomics useful?

Many microbes cannot be cultured alone in the lab — metagenomics lets scientists study them without isolation.

What are some uses of metagenomics?

Medicine, agriculture, bioremediation, biotechnology, global change studies, virus ID, and aquatic biology.

What is functional genomics?

Studying what genetic sequences do — gene function, gene product interactions, and metabolic pathways.

What is a DNA microarray used for?

Measuring the expression of thousands of genes simultaneously by hybridizing labeled cDNA to known DNA spots.

How does a DNA microarray work?

Isolate mRNA → make fluorescently labeled cDNA → hybridize to spotted array → scan: more fluorescence = more transcript.

What is ChIP?

Chromatin immunoprecipitation — uses antibodies to pull down proteins bound to DNA in living cells, then identifies the associated DNA.

What is RNA-Seq?

Sequencing cDNA made from RNA using next-generation methods to study the full transcriptome.

What is the transcriptome?

The complete set of RNA molecules transcribed in a cell or cell population.

What can RNA-Seq compare?

Different cell types, healthy vs. diseased cells, developmental stages, or responses to the environment.

What is proteomics?

The study of protein function and protein interactions across cells or organisms.

What is the proteome?

The complete set of proteins in a cell or organism.

Why is the proteome larger than the genome?

Because of alternative splicing, RNA editing, and post-translational covalent modifications.

What does 2D gel electrophoresis do?

Separates thousands of proteins — first by charge (isoelectric focusing), then by size (SDS-PAGE).

What does mass spectrometry do in proteomics?

Measures the molecular mass of peptide fragments to identify which protein corresponds to a gel spot.

What is an antibody microarray?

A protein array where antibodies are spotted; fluorescently labeled cellular proteins bind and intensity shows abundance.

What is a functional protein microarray?

Purified proteins are spotted and probed to test specific functions, such as phosphorylation by kinases.

What is bioinformatics?

Using computational, mathematical, and statistical tools to record, store, and analyze biological data.

What is the difference between cloning and PCR?

Cloning copies a gene inside a living host using a vector; PCR amplifies DNA in vitro using primers and polymerase.

What is the difference between cDNA and genomic DNA?

cDNA comes from mRNA (no introns); genomic DNA includes introns and regulatory regions.

Northern vs. Western vs. EMSA — what does each detect?

Northern = RNA; Western = protein; EMSA = protein binding to DNA or RNA.

Cytogenetic vs. linkage vs. physical mapping — key difference?

Cytogenetic uses a microscope; linkage uses recombination rates; physical uses cloned overlapping fragments.

Site-directed mutagenesis vs. CRISPR — key difference?

Site-directed mutagenesis makes a chosen base change for functional study; CRISPR makes double-strand cuts repaired by the cell.

DNA microarray vs. RNA-Seq — key difference?

Microarray hybridizes to known spots (limited to what's on the chip); RNA-Seq sequences all transcripts (unbiased).