TECHNIQUE
HOW IT WORKS
USE
CLUE
Cycles of heat denaturation (~95 °C), primer annealing (~55 °C), extension by heat-stable Taq polymerase (~72 °C)
Amplify a target sequence; diagnose infections, mutations, paternity
Gel electrophoresis then sizes the products
Reverse transcriptase makes cDNA from RNA, then PCR; real-time fluorescence quantifies
Viral load (HIV, HCV), BCR-ABL transcript monitoring, gene expression
Starts with RNA
Fragments migrate toward the + electrode; small fragments travel farther
Size DNA, RNA or protein
Trinucleotide repeat expansions → longer, slower bands
DNA cut by restriction enzymes → gel → membrane → labeled DNA probe
Detect DNA mutations, deletions, RFLPs
Southern = DNA
RNA on gel → membrane → DNA probe
Measure mRNA level / size
Northern = RNA
Protein on gel probed with labeled DNA
Find DNA-binding proteins (transcription factors)
—
Thousands of known probes on a chip; labeled patient DNA or cDNA hybridizes
Gene expression profiling, SNP genotyping, copy-number variants
Chromosomal microarray detects microdeletions a karyotype misses
Dideoxynucleotides (no 3′-OH) terminate chains at each base; read fragment lengths
Confirm a known mutation
Chain termination principle
Next-generation sequencing
Massively parallel reading of short fragments
Whole exome/genome, tumor panels, cell-free fetal DNA screening
Variants of uncertain significance are common
Guide RNA leads Cas9 nuclease to a matching sequence → double-strand break → repair by NHEJ (knock-out) or HDR (knock-in)
Gene editing (research, sickle cell therapy)
Derived from bacterial antiviral immunity
siRNA complementary to target mRNA → cleaved by RISC
Gene knockdown
Knock-down (↓ expression) vs knock-out (gene gone)
mRNA → cDNA (reverse transcriptase, no introns) → inserted into a plasmid → expressed in bacteria
Recombinant proteins (insulin)
Bacteria can't splice — use cDNA