CYCLE 10

Polymerase Chain Reaction (PCR)

A laboratory technique used to amplify specific DNA sequences, producing millions of copies.

PCR Basis

PCR is based on the natural process of DNA replication.

Primers

Short DNA sequences that bind to the target DNA and provide a starting point for DNA synthesis.

dNTPs

Deoxyribonucleotide triphosphates; the building blocks used by DNA polymerase to synthesize new DNA strands.

Taq Polymerase

A heat-stable DNA polymerase that synthesizes new DNA strands during PCR.

MgCl₂

Magnesium chloride; provides Mg²⁺ ions that act as essential cofactors for Taq polymerase.

Denaturation

The PCR step at ~95°C where double-stranded DNA separates into single strands.

Annealing

The PCR step at ~55°C where primers bind to complementary sequences on the DNA template.

Extension (Elongation)

The PCR step at ~72°C where Taq polymerase synthesizes new DNA strands.

Multiplex PCR

A variation of PCR that uses multiple primer sets to amplify several DNA sequences simultaneously.

Reverse Transcription (RT)

The process of converting mRNA into complementary DNA (cDNA) using reverse transcriptase.

cDNA

Complementary DNA synthesized from an mRNA template; contains only exons and represents expressed genes.

Reverse Transcriptase

An enzyme with DNA polymerase and RNase activities that synthesizes cDNA from mRNA.

Oligo(dT) Primer

A primer composed of thymine nucleotides used to bind the poly-A tail of mRNA during reverse transcription.

RNase Activity

The function of reverse transcriptase that degrades the RNA strand of an RNA-DNA hybrid.

Poly-A Tail

A sequence of adenine nucleotides at the 3' end of eukaryotic mRNA used for stability and primer binding.

Genomic DNA (gDNA)

The complete DNA of an organism, containing both exons and introns.

Short Tandem Repeats (STRs)

Highly polymorphic DNA sequences consisting of short repeating units used in DNA profiling.

CODIS

The FBI database that uses 13 STR loci for human identification.

Electrophoresis

A technique that separates DNA fragments based on size using an electric field.

Capillary Electrophoresis

A form of electrophoresis performed in a thin capillary tube, producing an electropherogram.

Electropherogram

A graphical output showing DNA fragment sizes and their relative abundance.

AMEL Gene

A gene used in DNA profiling to determine biological sex (XX = female, XY = male).

DNA Profiling

A forensic technique used to identify individuals based on STR patterns.

Plasmid

A small circular DNA molecule used as a vector to insert foreign genes into bacteria.

Transformation

The process of introducing recombinant plasmids into bacterial cells.

Recombinant Insulin Production

The process of producing human insulin in bacteria using cDNA inserted into plasmids.

CRISPR

A bacterial adaptive immune system used for genome editing; stands for Clustered Regularly Interspaced Short Palindromic Repeats.

PAM Sequence

A short DNA sequence (e.g., NGG) required for Cas9 recognition and binding.

Protospacer

A fragment of viral DNA inserted into the bacterial CRISPR locus during immunization.

CRISPR Locus

A region in the bacterial genome containing spacers from past viral infections separated by palindromic repeats.

pre-crRNA

The initial RNA transcript of the CRISPR locus containing multiple spacer sequences.

tracrRNA

Trans-activating CRISPR RNA that pairs with crRNA to guide Cas9.

crRNA

CRISPR RNA containing a spacer sequence that directs Cas9 to the target DNA.

Cas9

An endonuclease that creates double-stranded breaks in target DNA during CRISPR editing.

Ribonucleoprotein Complex

The functional complex formed by Cas9, crRNA, and tracrRNA that targets and cleaves DNA.

Non-Homologous End Joining (NHEJ)

An error-prone DNA repair mechanism that introduces insertions or deletions after a double-stranded break.

Homology-Directed Repair (HDR)

A precise DNA repair mechanism that uses a homologous template to repair double-stranded breaks.

Base Editing

A CRISPR-based technique that converts one nucleotide to another without creating a double-stranded break.

nCas (Nickase Cas9)

A modified Cas9 enzyme that introduces a single-strand nick instead of a double-stranded break.

Cytidine Deaminase

An enzyme used in base editing to convert cytosine (C) to uracil (U).

Uracil Glycosylase Inhibitor (UGI)

A protein that prevents the removal of uracil during base editing, enabling base conversion.

PD-1

A receptor on T-cells that suppresses immune responses and can be knocked out using CRISPR for cancer therapy.

T-Cell CRISPR Therapy

A treatment approach where PD-1 is knocked out in T-cells to enhance their ability to attack cancer cells.