Primer Design and Cloning Strategies in Synthetic Biology

GC Clamp

Two or more G/C nucleotides on primer's 3' end.

Melting Point (Tm)

Temperature where 50% of DNA strands are double-stranded.

Primer Length

Primers should be 20-25 nucleotides long.

Uniqueness

Primers must bind only once in DNA sequence.

High GC Content

Primers should have >60% GC for stability.

High AT Content

Primers can have >80% AT, affecting annealing.

Hairpin

Self-complementary structure in primers, causing issues.

Palindromes

Sequences that read the same forwards and backwards.

Slipping

Runs of identical nucleotides in primer sequences.

Transformation Efficiency

Circular plasmids transform better than linear ones.

Cytotoxicity

Toxic effects from certain DNA sequences or proteins.

Site-Directed Mutagenesis

Introducing specific mutations at defined positions.

Deletions

Removing specific amino acids or nucleotides from sequences.

Insertions

Adding specific amino acids or nucleotides to sequences.

DPN1 Enzyme

Digests methylated DNA, removing template plasmid.

T4 Ligase

Enzyme that ligates DNA fragments together.

PNK Enzyme

Phosphorylates DNA ends for ligation.

Complementary Strands

Strands that pair according to base pairing rules.

Forward Primer

Matches target DNA sequence in 5' to 3' direction.

Reverse Primer

Complementary to the target DNA, designed in reverse.

Reverse Complement

Complement of the reverse strand for primer design.

Substitution Primer Design

Mutation placed at 5' end of forward primer.

Overhang

Extra nucleotides added before mutation in primers.

E→G mutation

A specific nucleotide change from E to G.

ADE mutation

A specific deletion mutation affecting adenine.

CAT mutation

An insertion of CAT sequence into DNA.

FAT CAT mutation

A large insertion mutation of ≥10 nucleotides.

Chimeras

Fusion of different proteins not found naturally.

Fusions

Concatenation of two proteins into one.

Gibson Assembly

Technique using nuclease for DNA assembly.

Gibson Primer Design

Primers designed with ~25-50 nucleotide overlap.

Gblocks

Synthetic inserts used in genetic engineering.

Codon

Triplet of nucleotides coding for an amino acid.

tRNA

Transfer RNA carrying amino acids to ribosomes.

Anticodon

tRNA sequence that base pairs with mRNA codon.

A site

Ribosomal site where tRNA first binds.

P site

Ribosomal site for amino acid polymerization.

E site

Ribosomal site where tRNA exits.

Translation

Process of synthesizing proteins from mRNA.

Transcription

Synthesis of mRNA from a DNA template.

Reverse Transcription

Conversion of RNA back to DNA.

Frameshifts

Mutations causing misreading of codons.

Single Nucleotide Polymorphisms (SNPs)

Variations at a single nucleotide position.

Monocistronic genes

Genes coding for a single protein.

Polycistronic genes

Genes coding for multiple proteins.

Central Dogma

Flow of genetic information: DNA → RNA → Protein.

Wobble Position

Last anticodon nucleotide pairs flexibly with mRNA.

Codon Degeneracy

Multiple codons can specify the same amino acid.

Start Codon

AUG; initiates protein synthesis with methionine.

Stop Codons

Signals termination of protein synthesis; varies by organism. UAA, UAG, and UGA

Frameshift Mutation

Nucleotide shift alters downstream translation reading frame.

+1 Frameshift

One nucleotide addition shifts reading frame by one.

+2 Frameshift

Two nucleotide addition shifts reading frame by two.

Single Nucleotide Polymorphism (SNP)

Single nucleotide change; may or may not alter amino acid.

Silent Mutation

No amino acid change despite nucleotide alteration.

Point Mutation

Single nucleotide change results in different amino acid.

Insertions

Addition of nucleotides; can cause frameshifts if not in multiples of three.

Deletions

Removal of nucleotides; can cause frameshifts if not in multiples of three.

In-Frame Deletion

Deletion maintains reading frame; fewer amino acids produced.

Out-of-Frame Insertion

Insertion disrupts reading frame; alters downstream translation.

Monocistronic Genes

One gene codes for one protein.

Polycistronic Genes

One gene codes for multiple proteins.

Non-Coding DNA

DNA that does not code for proteins; often regulatory.

Human Genome Project

Initiative to map all human genes and their functions.

CRISPR-Cas9

Bacterial immune system used for gene editing.

NHEJ Pathway

Non-Homologous End Joining; DNA repair mechanism.

HR Pathway

Homologous Recombination; DNA repair using homologous sequences.

GMO Definition

Genetically Modified Organism; lab-altered organisms.

Dolly the Sheep

First cloned mammal; significant in genetic research.

Cas9

Endonuclease that creates double-stranded DNA breaks.

CRISPR

DNA sequences targeted by Cas9 in gene editing.

gRNA

Guide RNA processed from DNA sequences for targeting.

HR

Homologous recombination; uses templates for DNA repair.

NHEJ

Non-homologous end joining; ligates DNA without templates.

Rad51

Protein involved in homologous recombination repair.

BRCA1/2

Genes linked to DNA repair and cancer risk.

TdT

Terminal deoxynucleotidyl transferase; involved in NHEJ.

Artemis

Nuclease that processes DNA ends in NHEJ.

Cas12a

Endonuclease with simpler RNA targeting and sticky ends.

Cas13

Targets RNA instead of DNA for gene regulation.

Casgevy

FDA approved therapy for Sickle Cell Anemia.

Point mutants

Genetic mutations causing specific diseases.

Cystic Fibrosis

Disease affecting mucus membranes due to mutations.

Tay-Sachs Disease

Neurodegenerative disorder affecting brain and spinal cord.

Down syndrome

Genetic disorder causing developmental delays and abnormalities.

Spinal muscular atrophy

Progressive muscle wasting due to genetic mutations.

Crohn's disease

Chronic inflammatory bowel disease with severe symptoms.

Polycystic kidney disease

Genetic disorder leading to kidney failure.

ALS

Neurodegenerative disease with multiple genetic causes.

Knockout mouse

Mouse with a nonfunctional gene for research.

Knock-in

Modification of a gene, often with GFP.

Electroporation

Technique to introduce DNA into cells using electricity.

Blastocyst

Stage of embryo development for stem cell injection.

Homozygous population

Genetically uniform group with specific gene modifications.

Global Alignment

Aligns entire sequences from start to end.

Local Alignment

Aligns specific regions within sequences.

Pairwise Alignment

Compares two sequences for similarity.

BLAST

Basic Local Alignment Search Tool for sequence comparison.

Multiple Sequence Alignment (MSA)

Aligns three or more sequences simultaneously.

ClustalW

Progressive alignment tool for multiple sequences.