13-14 Nov

Expose membrane to UV

Crosslinks DNA to blot

Incubate with radioactive probe

Hybridizes to complementary DNA sequences

Wash blot

Removes excess probe

Expose to X-ray film

Produces bands where the probe annealed

PCR amplification

Relies on temperature changes for DNA denaturation, primer annealing, and polymerization

PCR requirements

Template, primers, dNTPs, DNA polymerase

Reverse transcription PCR

RNA converted to cDNA, then amplified

DNA polymorphisms

Include SNPs, STRs, VNTRs

SNP detection

By restriction enzymes or microarrays

STR detection

Amplified by PCR with flanking primers

VNTR detection

Southern blotting and restriction enzymes

DNA typing

Used in forensics, paternity testing, and conservation biology

Gene therapy

Technique for correcting defective genes

Vectors in gene therapy

Include retroviruses, adenoviruses, and AAVs

CRISPR-Cas9

Genome-editing tool for precise DNA modification

TALENs and ZFNs

Alternatives for targeted genome editing

lac operon regulation

Controlled by glucose and lactose availability

AraBAD operon

Induced by arabinose, regulated by AraC protein

Trp operon regulation

Repressed by tryptophan uses attenuation

Enhancesome

Multimeric complex increasing transcription levels

Helix-turn-helix motif

Found in DNA-binding proteins

Combinatorial gene regulation

Controlled by combinations of activators

GAL genes in yeast

Activated in the presence of galactose

Steroid hormones

Regulate gene expression in mammalian cells

miRNA and siRNA

Silence genes post-transcriptionally via RISC

p53 protein

Key role in cancer prevention by binding DNA

Tumor classification

Benign (localized) vs malignant (invasive)

Metastasis

Spread of malignant cells to other body parts

Gene silencing

Can be achieved through heterochromatin formation

Histone acetylation

Opens chromatin for transcriptional activation

RNA Pol II

Works with basal transcription factors to transcribe genes

Transcription activators

Bind enhancers to increase gene expression

Mediator complex

Bridges activators and RNA Pol II

DNA looping

Brings distant regulatory elements close to promoters

Non-coding RNAs

Include miRNA, siRNA, and lncRNA for regulation

RFLP analysis

Detects genetic mutations or polymorphisms

PCR with ASO hybridization

Used for specific allele detection

Gene expression control

Occurs at transcriptional and post-transcriptional levels

CRP gene

Encodes CAP for lac operon positive regulation

lacI repressor

Blocks RNA Pol unless lactose is present

LacOc mutation

Prevents repressor binding; constitutive expression

AraC protein

Acts as repressor or activator based on arabinose presence

Chromatin remodeling

Alters nucleosome position for transcription

PaJaMo experiment

Confirmed lacI as negative regulator of lac operon

Proteins in trans

Diffuse and act on multiple DNA molecules

DNA elements in cis

Influence only adjacent genes

Eukaryotic RNA polymerases

Pol I (rRNA), Pol II (mRNA), Pol III (tRNA)

Gene mutations in cancer

p53 and tumor suppressor genes implicated

Gene silencing via RISC

miRNA inhibits translation or cleaves mRNA

Steroid hormone receptors

Regulate transcription through binding DNA

BRCA1/BRCA2 mutations

Increase risk of breast and ovarian cancers

tumor microenviorment

Influences cancer progression and metastasis

DNA looping in regulation

Facilitates interactions between distant regions

trpR- strains

Show higher transcription without tryptophan

Gene therapy challenges

Include immune response and short-lived effects

FDA on gene therapy

Experimental with some recent successes

First blindness gene therapy trial

Improved vision in LCA patients

Gene editing for Tay-Sachs

Uses CRISPR for potential cures

Gene regulation in cancer

Altered pathways can drive malignancy

Activator motifs

Include leucine zippers and zinc fingers

Mediator’s role

Coordinates transcription machinery assembly

Trp operon attenuation

Depends on mRNA structure

CAP-cAMP in lac operon

Positive control under low glucose

GAL system in yeast

GAL system in yeast

DNA-binding domains

Essential for transcription factor function

Metastatic progression

Hallmark of advanced cancers

Chromatin structure’s role

Key in eukaryotic gene regulation

Trans-splicing in C. elegans

Produces monogenic mRNAs

Regulation by enhancers

Requires activators and coactivators

DNA silencing mechanisms

Involve histone modification and non-coding RNAs