Step 1 · Biochemistry

Lab Techniques: PCR, Blotting, CRISPR

Name: Lab Techniques: PCR, Blotting, CRISPR
Creator: Veli Bakalov, MD

Amplify, probe, sort, and edit nucleic acids

Biochemistry·Molecular Biology·5 min read·9 high-yield points·Reviewed by Veli Bakalov, MD @HemeOncBuddy

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PCR needs template, two flanking primers, dNTPs, and heat-stable Taq (Thermus aquaticus); cycles of denaturation ~95 °C, annealing ~55 °C, and extension ~72 °C amplify DNA exponentially (2^n).
RT-PCR converts RNA to cDNA first (RNA viruses such as SARS-CoV-2, HIV, HCV); quantitative real-time PCR reads fluorescence each cycle to measure viral load.
SNoW DRoP: Southern = DNA, Northern = RNA, Western = protein; Southern and Northern use nucleic-acid probes, Western uses antibodies.
Southern blot sizes DNA and detects gene rearrangements and trinucleotide repeat expansions (fragile X CGG, Huntington CAG).
ELISA (antibody-enzyme color change) is the sensitive screening test (HIV); Western blot confirms a specific protein, as in Lyme disease after a positive ELISA.
Flow cytometry immunophenotypes leukemias and lymphomas, counts CD4 cells, and diagnoses PNH by absent CD55 and CD59.
FISH detects microdeletions (DiGeorge 22q11), translocations (BCR-ABL t(9;22)), and HER2 amplification that a karyotype misses (resolution about 5 Mb).
Karyotype (metaphase chromosomes) detects aneuploidy and large rearrangements; SNP microarrays find genome-wide copy-number changes and genotype variants for GWAS.
CRISPR/Cas9: a guide RNA directs Cas9 to make a double-strand break next to a PAM; NHEJ repair knocks a gene out, HDR knocks a sequence in.

PCR and its variants

Ingredients. The polymerase chain reaction copies a chosen DNA segment in a tube. It needs the template DNA, two primers that flank the target, a heat-stable DNA polymerase (Taq), and deoxynucleotides (dNTPs).

Denaturation, ~95 °CHeat separates the two DNA strands
Annealing, ~55 °CPrimers bind their complementary template sites
Extension, ~72 °CTaq synthesizes the new strands from the primers
Repeat 20–40 cyclesEach cycle doubles the target: exponential amplification

One PCR cycle

Why Taq. Taq polymerase comes from Thermus aquaticus, a hot-spring bacterium, so it survives the repeated ~95 °C denaturation steps. It lacks proofreading activity, so it is chosen for heat stability, not fidelity.

2ⁿCopies per starting molecule after n cycles

~10⁹Fold amplification after 30 cycles

Reading the result. Products are usually visualized by gel electrophoresis, which separates DNA fragments by size.

RT-PCR. Reverse transcriptase first copies RNA (e.g., mRNA) into complementary DNA (cDNA), which PCR then amplifies. This is how RNA viruses such as SARS-CoV-2, HIV, and HCV are detected.

Quantitative (real-time) PCR. Fluorescence is measured every cycle; the sooner the signal rises, the more starting template was present. It quantifies nucleic acid, as in viral load testing or monitoring BCR-ABL transcripts in CML.

Do not mix up the abbreviations: RT-PCR (reverse transcription) lets you amplify RNA; real-time/quantitative PCR measures how much is there. The two are often combined.

Blotting: SNoW DRoP

General scheme. Molecules are separated by size on a gel, transferred ("blotted") to a membrane, and detected with a labeled probe. Mnemonic SNoW DRoP: Southern = DNA, Northern = RNA, Western = Protein.

The three blots

Blot	Detects	Probe	Classic uses
Southern	DNA (restriction-cut fragments)	Labeled DNA probe	Gene rearrangements; trinucleotide repeat expansions (Huntington CAG, fragile X CGG)
Northern	RNA	Labeled nucleic-acid probe	Sizes and quantifies mRNA: whether and how much a gene is expressed in a tissue
Western	Protein (separated by SDS-PAGE)	Antibody	Lyme disease confirmation after a positive ELISA; historically, HIV confirmation

The Southwestern blot uses labeled oligonucleotide probes to find DNA-binding proteins such as transcription factors.

Southern blot for repeat disorders. Because it directly sizes large DNA fragments, and can be paired with methylation-sensitive restriction enzymes, the Southern blot is the classic way to confirm a fragile X full mutation of several hundred CGG repeats with abnormal methylation. The Northern blot is simply its RNA analog.

Antibody-based assays: ELISA and flow cytometry

ELISA. An antibody linked to an enzyme produces a color change when its target is present; the assay can detect either an antigen or an antibody in a sample. Being highly sensitive, it serves as the HIV screening test and detects many autoantibodies.

Flow cytometry. Cells tagged with fluorophore-linked antibodies pass one by one through a laser: forward scatter reflects size, side scatter reflects granularity, and fluorescence identifies surface markers. Uses include immunophenotyping leukemias and lymphomas, CD4 counts in HIV, and diagnosing paroxysmal nocturnal hemoglobinuria (PNH) by the absence of the GPI-anchored proteins CD55 and CD59.

Antibody-based tests

Feature	ELISA	Western blot	Flow cytometry
Sample	Fluid; detects antigen or antibody	Proteins separated by SDS-PAGE	Intact cells in suspension
Readout	Enzyme-driven color change	Antibody-labeled bands	Fluorescence and light scatter per cell
Typical role	Sensitive screening (HIV)	Confirms a specific protein (Lyme)	Immunophenotyping, CD4 count, PNH

Cytogenetics: karyotype, FISH, microarrays

Karyotype. Dividing cells are arrested in metaphase, stained, and their chromosomes ordered by size and banding pattern. It detects aneuploidy (trisomy 21), large deletions, and translocations but cannot resolve microdeletions smaller than about 5 Mb.

FISH. Fluorescence in situ hybridization binds a fluorescent DNA probe to one chosen chromosomal region. It reveals microdeletions (DiGeorge 22q11), translocations (BCR-ABL, t(9;22)), and gene amplification (HER2) — changes too small for a karyotype — and works on nondividing (interphase) cells.

DNA microarrays. Sample nucleic acid hybridizes to thousands of chip-bound probes. Expression arrays profile mRNA to classify tumors; SNP arrays genotype variants for genome-wide association studies (GWAS) and detect copy-number changes and microdeletions across the whole genome. Balanced rearrangements, which do not change copy number, are missed.

Choosing a chromosome test

Feature	Karyotype	FISH	SNP / chromosomal microarray
Scope	Whole genome, low resolution	One targeted locus	Whole genome, high resolution
Best for	Aneuploidy, large rearrangements	Known microdeletion, translocation, amplification	Copy-number changes anywhere; genotyping
Misses	Deletions under ~5 Mb	Anything outside the probe	Balanced translocations

Suspected microdeletion — for example, a child with a conotruncal heart defect, hypocalcemia, and recurrent infections (DiGeorge, 22q11.2): order FISH or a chromosomal microarray, not a standard karyotype.

Genome editing with CRISPR/Cas9

Mechanism. A guide RNA base-pairs with the matching genomic sequence and directs the Cas9 nuclease there; Cas9 cuts only where the target lies next to a short PAM sequence. The guide RNA supplies specificity; Cas9 is the molecular scissors.

TargetingGuide RNA pairs with the chosen DNA sequence
CuttingCas9 makes a double-strand break beside the PAM
Repair by NHEJError-prone joining leaves small insertions or deletions: gene knockout
Repair by HDRA supplied donor template is copied in: precise knock-in

CRISPR/Cas9 editing

Clinical use. The same double-strand break repair pathways used by cells after DNA damage are harnessed here. Ex vivo editing of a patient's own hematopoietic stem cells underlies gene-editing therapy for sickle cell disease, which works by reactivating fetal hemoglobin production.