22.3 Molecular biology techniques in cell research

PCR and recombinant DNA techniques are game-changers in cell biology. They let scientists copy, modify, and study DNA with incredible precision. These tools have revolutionized everything from gene discovery to protein production.

Gene silencing and expression analysis take things even further. By turning genes off or measuring their activity, researchers can unravel complex cellular processes. It's like having a molecular Swiss Army knife for cell biology experiments.

Polymerase Chain Reaction (PCR) and DNA Amplification

Principles of PCR amplification

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• PCR amplifies specific DNA sequences by producing millions of copies of a target DNA sequence from a small initial sample
• Requires several components including DNA template with target sequence, two primers complementary to 5' ends of target sequence on each strand, DNA polymerase (Taq polymerase) for DNA synthesis, deoxynucleoside triphosphates (dNTPs) as building blocks for new DNA strands, and buffer to maintain optimal conditions for DNA polymerase
• Involves three main steps repeated in cycles:
  1. Denaturation: High temperature (94-96°C) separates double-stranded DNA template into single strands
  2. Annealing: Lower temperature (50-65°C) allows primers to bind to complementary sequences on single-stranded DNA
  3. Extension: Temperature raised (72°C) to optimize DNA polymerase activity, synthesizing new DNA strands complementary to template
• Number of target DNA sequences doubles with each cycle resulting in exponential amplification

Applications of PCR in cell biology research

• Amplifies specific genes or DNA sequences for cloning or sequencing (gene isolation, genetic engineering)
• Detects presence of specific DNA sequences for genotyping or identifying pathogens (diagnostic testing, forensic analysis)
• Quantifies amount of a specific DNA sequence in a sample using quantitative PCR or qPCR (gene expression analysis, viral load monitoring)

Recombinant DNA Technology

Techniques for recombinant DNA construction

• Restriction enzymes cut DNA at specific recognition sequences resulting in blunt ends or sticky ends with short overhangs (EcoRI, BamHI)
• DNA ligation joins two DNA fragments together catalyzed by DNA ligase forming a phosphodiester bond between 3' hydroxyl group of one fragment and 5' phosphate group of another, with sticky ends facilitating ligation of specific fragments
• Cloning inserts a DNA fragment of interest into a vector that can replicate independently of host genome (plasmids, viral vectors), with vector and fragment cut by same restriction enzyme(s) to generate compatible ends for ligation, then introduced into host cell (bacteria, eukaryotic cells) for replication and expression

Applications of recombinant DNA in cell biology research

• Produces large quantities of specific proteins by cloning corresponding gene into an expression vector (insulin production, enzyme manufacturing)
• Generates transgenic organisms or cell lines with altered gene expression for functional studies (knockout mice, genetically modified crops)
• Studies effects of specific mutations or modifications on gene function by introducing engineered DNA sequences into cells (site-directed mutagenesis, reporter gene assays)

Gene Silencing and Genome Editing

Applications of RNAi and CRISPR-Cas9

• RNA interference (RNAi) inhibits gene expression by targeting specific mRNAs for degradation using small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) incorporated into RNA-induced silencing complex (RISC) that cleaves complementary mRNA sequences
  • Studies effects of gene knockdown on cellular processes and identifies gene functions (loss-of-function screens, pathway analysis)
• CRISPR-Cas9 is a genome editing tool adapted from bacterial immune system using guide RNA (gRNA) to direct Cas9 endonuclease to create double-strand breaks at specific genome locations, with cell's DNA repair mechanisms (non-homologous end joining or homology-directed repair) harnessed to introduce specific mutations or insertions at target site
  • Generates knockout cell lines or organisms to study effects of gene loss on cellular processes (disease modeling, functional genomics)
  • Introduces specific mutations or modifications into genes to study their function (knock-in models, precise gene editing)
  • Performs genome-wide screens to identify genes involved in specific biological processes (drug target discovery, genetic interactions)

Gene Expression Analysis

Methods for gene expression analysis

• RNA isolation purifies RNA from biological samples by lysing cells or tissues in presence of RNase inhibitors, separating RNA from DNA and proteins using phenol-chloroform extraction or column-based purification kits, and treating with DNase to remove genomic DNA contamination
• Reverse transcription synthesizes complementary DNA (cDNA) from RNA template using reverse transcriptase (RNA-dependent DNA polymerase) with oligo(dT) primers that anneal to poly(A) tails of eukaryotic mRNAs or random hexamer primers, producing cDNA for subsequent PCR amplification or other downstream applications
• Quantitative real-time PCR (qRT-PCR) measures abundance of specific RNA transcripts using cDNA from reverse transcription as template, monitoring amplification of target sequence in real-time with fluorescent dyes or probes, and comparing amplification curves of target gene to reference gene (housekeeping gene) to quantify relative gene expression levels
  • Compares gene expression levels between different samples or conditions (treated vs untreated, diseased vs healthy)
  • Validates results obtained from other gene expression analysis methods like microarrays or RNA sequencing
  • Studies effects of various treatments or perturbations on gene expression (drug response, environmental stress)