Genetic Engineering & Molecular Biology Techniques: DNA, RNA, and Protein Analysis

Forward genetics

Identifies mutants with a phenotype, then determines the responsible gene.

Reverse genetics

Alters a known gene to observe the resulting phenotypic effect.

Recombinant human insulin example

Human insulin gene cloned into bacteria to produce insulin mRNA and protein; first approved recombinant therapeutic (1982).

Pre-1980s insulin source

Insulin was purified from bovine or porcine pancreas before recombinant methods.

Insulin function

Peptide hormone regulating glucose, carbohydrate, lipid, and protein metabolism.

Restriction enzymes

Restriction endonucleases cut DNA at specific palindromic recognition sequences.

Sticky ends

Staggered DNA cuts producing single-stranded overhangs that can base-pair with complementary ends.

Blunt ends

Straight DNA cuts leaving no overhangs.

Bacterial restriction enzymes

Protect bacteria from viral (foreign) DNA by degrading it.

Southern blot

Detects specific DNA sequences using labeled probes after gel electrophoresis.

Northern blot

Detects and quantifies RNA molecules.

Western blot

Detects proteins using specific antibodies.

PCR purpose

Amplifies DNA fragments in vitro.

RT-PCR

Reverse-transcribes RNA to cDNA before amplification, allowing RNA quantification.

In situ hybridization (ISH)

Uses radioactive probes to localize nucleic acids in cells or tissues; detected via autoradiography.

FISH

Fluorescent in situ hybridization; uses fluorescent probes for DNA/RNA visualization in cells.

Immunofluorescence

Uses fluorescently labeled antibodies to visualize specific proteins in cells.

Agarose gel electrophoresis

Separates DNA, RNA, or proteins by size and charge.

Electrophoresis direction

Molecules migrate from cathode (-) to anode (+).

Visualization of DNA/RNA

Ethidium bromide staining under UV light reveals nucleic acids.

Protein visualization

Coomassie Blue dye stains proteins in gels.

Southern/Northern probe type

Radioactive ³²P single-stranded DNA or RNA complementary to the target sequence.

Western probe type

Specific antibody detected by chemiluminescence or fluorescence.

Blot transfer method

Gels soaked in NaOH to denature DNA; DNA transfers to membranes by capillary action.

Insulin gene comparison

Human, mouse, and rat insulin genes share similar sequences and products; located on chromosomes 11, 19, 7 respectively.

Restriction digestion example

EcoRI produces a 13 kb fragment, XhoI produces a 5 kb fragment containing the insulin gene.

Blot probe example

Probe 1 targets exon 2 of Ins gene; RpS7 used as a control probe.

Insulin protein size

Approximately 12 kDa.

PCR full name

Polymerase Chain Reaction.

PCR components

Template DNA, primers, Taq polymerase, dNTPs, buffer, thermocycler.

Taq polymerase origin

Thermus aquaticus; optimal 75-80 °C; heat-stable.

PCR steps

Denaturation (95 °C) → Annealing (~60 °C) → Extension (72 °C).

PCR purpose

Exponential amplification of specific DNA fragments through repeated cycles.

Reverse transcription (RT)

Reverse transcriptase converts mRNA → ssDNA; DNA polymerase I synthesizes complementary strand to form ds cDNA.

Generating recombinant DNA

Combines DNA fragments from different sources into a single molecule.

Restriction + ligation process

Restriction enzymes cut both vector and insert; DNA ligase seals them together.

Donor DNA sources

Genomic digests, PCR products, or cDNA from mRNA.

Vector requirements

Restriction sites, selectable marker, and capacity for replication/expression.

Plasmid vectors

Small circular DNAs replicating independently in bacteria.

Essential plasmid features

Origin of replication (ori), antibiotic resistance gene, lacZ screening gene, multiple cloning site (MCS).

Example vector pUC18

Contains ampᴿ and lacZ genes with MCS for insertion.

Blue-white screening

Insertion into lacZ disrupts β-galactosidase; white colonies = recombinant; blue = non-recombinant.

Other vector types

Bacteriophage λ, fosmids, and BACs are used for larger DNA inserts.

Bacteriophage λ capacity

Accepts ≈15 kb DNA insert by replacing central region.

Fosmids

λ phage × F plasmid hybrids that replicate as plasmids.

BACs (Bacterial Artificial Chromosomes)

F plasmid derivatives carrying large (>100 kb) inserts.

Vector delivery methods

Transformation (plasmids/BACs), transduction (fosmids), infection (phage λ).

Vector recovery

Phage lysate collection or bacterial lysis + DNA purification by centrifugation/electrophoresis.

Genomic library definition

Collection of recombinant clones representing an entire genome.

Human genome coverage example

3 × 10⁹ bp requires ~375 000 clones (40 kb each) for 5× coverage using fosmids.

Identifying clones

Labeled DNA/RNA probes hybridize to target gene within library.

Expression library screening

Uses antibodies to detect colonies expressing desired protein.

Genomic vs cDNA libraries

cDNA = no introns, polyA tail; used for protein expression; genomic = introns + regulatory sequences.

Sanger sequencing principle

Incorporates dideoxynucleotides (ddNTPs) lacking 3′-OH to terminate chain elongation.

Number of reactions in classic method

Four reactions (1 per base type) separated by polyacrylamide gel electrophoresis.

Band pattern interpretation

Each lane differs by 1 nucleotide; sequence read from short to long fragments.

Automated sequencing

Uses fluorescently labeled ddNTPs detected by laser and computer software.

Fluorescent color coding

A = green, C = blue, G = black, T = red; "N" = overlap or uncertain base call.

Genetic engineering overview

Modifying genomes to introduce new traits or produce useful products.

Traditional vs modern methods

Selective breeding (older) vs direct DNA modification (modern).

GE crop statistics (US 2020)

92 % corn, 94 % soybeans, 96 % cotton genetically engineered.

Transgenic examples

Atlantic salmon (overexpress GH), GloFish, goats producing human antithrombin, "Golden Rice," antifreeze plants.

Transgene definition

Transferred gene introduced into another organism.

Transgenic organism

Organism expressing a foreign (transgene) gene.

Biotechnology definition

Practical use of genetic engineering in industry, medicine, and agriculture.

Chemical DNA introduction

DNA precipitated with Ca-phosphate or in lipid vesicles → endocytosis into cells.

Physical methods of gene delivery

Electroporation (electric pulses create membrane pores), gene gun (DNA-coated particles), microinjection (fine needle).

Biological delivery methods

Agrobacterium tumefaciens (T-DNA transfer via Ti plasmid) and viral vectors for animal cells.

Transgene fate in nucleus

Integration by homologous recombination or ectopic insertion, or episomal replication.

Ti plasmid role in plants

Modified to deliver genes to plants without causing disease.

Plant transformation procedure

Infect tissue (e.g., tobacco leaf disks) with engineered Agrobacterium; select on antibiotic medium.

Callus formation

Transformed cells form calli that regenerate into whole transgenic plants.

CRISPR-Cas9 origin

Adapted from bacterial immune system (Streptococcus pyogenes).

CRISPR mechanism in bacteria

Stores viral DNA spacers in CRISPR arrays to recognize future infections.

Cas9 function

Endonuclease with two nuclease domains that cut each DNA strand.

PAM sequence requirement

NGG motif required for Cas9 binding and cleavage.

gRNA and tracrRNA roles

gRNA guides Cas9 to target; tracrRNA stabilizes complex.

sgRNA definition

Single-guide RNA = fusion of gRNA and tracrRNA into one molecule.

CRISPR developers

Emmanuelle Charpentier and Jennifer Doudna (Nobel Prize 2020).

System components

Plasmid 1 = Cas9, Plasmid 2 = sgRNA with custom target, optional donor template for repair.

Repair pathways after cleavage

NHEJ = error-prone indels → gene inactivation; HR = precise repair via donor DNA.

CRISPR application example 1

Two sgRNAs flank mouse Ins1 gene → NHEJ deletion mutant.

CRISPR application example 2

Donor plasmid replaces Ins1 with GFP → β-cells fluorescent.

Overview

Methods used to study the function of one or a few genes within a genome involve detecting, quantifying, and altering DNA, RNA, or protein.