Laboratory methods

The laboratory methods explained here are used to screen for and confirm medical conditions. For additional laboratory methods see “Pathology techniques.”

Preanalytical phase

The preanalytical phase encompasses the selection of relevant diagnostic tests and the collection and transport of samples. Specificity and sensitivity are important factors that should be considered when selecting a diagnostic test (e.g., many screening tests have a high sensitivity but low specificity). See also “Evaluation of diagnostic tests” in “Epidemiology.”

• Commonly analyzed biofluids
  • Blood
  • Urine (see “Diagnostic evaluation of the kidney and urinary tract”)
  • Cerebrospinal fluid (see “Cerebrospinal fluid analysis”)
  • Fluid collection from punctures (e.g., ascites, pleural effusion)
• Interference factors: affect sample quality
  • Errors during sample collection (e.g., prolonged venous stasis during blood collection, which can lead to falsely elevated levels of potassium and calcium )
  • Errors during sample transport, such as:
    • Exposure to strong light (e.g., leading to degradation of bilirubin, resulting in falsely low levels)
    • Shaking of blood samples, leading to hemolysis

Preanalytical-phase errors (i.e., errors during sample collection or transport) should be considered if actual test results differ significantly from expected results.

Analytical phase

The analytical phase comprises sample processing and the generation of results.

• Qualitative methods: The result is either positive or negative. Examples include:
  • Pregnancy test
  • Rapid antigen detection test
  • Urine dipstick
• Quantitative methods: The concentration of a substance is determined. Examples include:
  • Immunochemistry: e.g., ELISA for the detection of PSA
  • Photometry: e.g., indirect determination of glucose concentration
  • Microscopy: e.g., manual blood cell count with differential

Postanalytical phase

The most important steps of the postanalytical phase include:

1. Ensuring the correct allocation of results to patient data
2. Plausibility check
3. Conveyance of results
4. Medical interpretation based on the combination of test results, patient history, and physical examination findings

Results from laboratory studies should always be interpreted in conjunction with the medical history, clinical examination, and other diagnostic tests.

• A technique used to detect DNA, RNA, and proteins that involves transferring the DNA, RNA, or proteins onto a membrane. The sample of interest is then visualized using marked detector molecules (e.g., radiolabeled DNA or chemiluminescent detector RNA).
• The blotting techniques most commonly employed in laboratory medicine are Southern, western, and northern blot.
• All blotting techniques follow a similar process:
  1. DNA, RNA, or protein molecules contained in the sample are separated by gel electrophoresis and/or electric charge, depending on their size (small molecules travel faster than bigger molecules).
  2. The separated molecules are transferred from the gel onto a membrane.
  3. The DNA, RNA, or protein molecule of interest is detected by using a labeled, highly specific oligonucleotide probes, antibodies, or x-rays.

SNoW flakes DRoP: Southern blot, Northern blot, and Western blot are used on DNA, RNA, and Proteins respectively.

• Sample: : RNA
• Principle of detection: annealing of marked detector RNA or DNA to the target RNA fragment
  • ³²P-DNA or -RNA
  • DNA or RNA labeled with a chemiluminescent dye
• Procedure
  • The RNA sample is cleaved by enzymes and separated by gel electrophoresis (commonly on agarose gels).
  • Separated and cleaved RNA is transferred (blotted) to a membrane.
  • The membrane is incubated with labeled probes of RNA or DNA.
  • Labeled probes recognize and anneal to the complementary strand if it is present on the membrane.
• Result: double-stranded RNA
  • One unlabeled strand (cleaved RNA sample)
  • One labeled strand that can be visualized using one of the following techniques:
    • When radiolabeled detector DNA/RNA is used, an x-ray film is placed against the western blot. This film develops when exposed to the label, creating dark regions that correspond to the target protein band.
    • In case of chemiluminescent DNA/RNA, a CCD camera that captures a digital image is used.
• Uses
  • Assessment of gene expression by measuring mRNA levels (e.g., expression of the FMR1 gene in different tissues in fragile X syndrome)
  • Assessment of RNA splicing (including splicing errors)

• Sample: DNA (DNA restriction fragments)
• Principle of detection: annealing of marked detector RNA or DNA to the target DNA fragment
  • ³²P-DNA or -RNA
  • DNA or RNA labeled with a chemiluminescent dye
• Procedure
  • The DNA sample is cleaved by restriction enzymes.
  • DNA fragments are separated by gel electrophoresis.
  • Separated DNA fragments are transferred (blotted) to a filter membrane.
  • The membrane is exposed to (radio)labeled oligonucleotides: DNA probes recognize and anneal to the complementary strand if it is present on the membrane.
• Result: double-stranded DNA
  • One unlabeled strand (cleaved DNA sample)
  • One labeled strand that can be visualized when the membrane is exposed to x-ray film or digital imaging.
• Uses: detection of specific genes and nucleotide sequences
  • Restriction fragment length polymorphisms (RFLPs)
  • Minisatellite DNA and microsatellite DNA (called “variable number of tandem repeats,” VNTRs)

• Sample: proteins
• Principle of detection: Monoclonal antibodies (immunoglobulins that bind to a single, specific antigen) are used for the direct or indirect detection of proteins of interest.
  • Enzyme- or fluorophore-conjugated antibodies
  • Radiolabeled antibodies (¹²⁵I)
• Procedure
  • The protein sample is separated by gel electrophoresis.
  • Separated proteins are transferred (blotted) to a membrane.
  • The protein of interest is detected by a specific antibody that can be labeled directly or detected by a secondary, radiolabeled antibody.
• Uses
  • Measuring the amount of antigens or antibodies
  • Confirmation of the presence of a protein in a sample (e.g., confirmatory test for antibodies against Borrelia in the diagnosis of Lyme disease)
  • Rough estimation of the amount of protein in a sample

• Sample: DNA-binding proteins
• Principle of detection: target proteins binding to radiolabeled double-stranded DNA probes
• Procedure ^[1]
  • Proteins are separated by gel electrophoresis and blotted onto a nitrocellulose membrane (similar to western blot).
  • Target proteins bind the radiolabeled double-stranded DNA probes (similar to Southern blot).
• Uses: study of DNA-binding proteins (e.g., transcription factors, such as c-Fos, c-Jun)

References: ^[2]

Overview

• ELISA is an enzyme immunoassay that employs enzyme-labeled immunoreactants and an immunoadsorbent to determine the presence and concentration of certain proteins (e.g., tumor markers, viral proteins, drugs, antibodies) in serum
• The detection method is based on the highly specific immunologic interaction between an antibody and its antigen (i.e., the protein of interest).
  1. The antigen is fixed on a microtiter plate and bound by an enzyme-coupled antibody.
  2. The enzyme catalyzes a reaction when incubated with its substrate which is chromogenic, chemifluorescent, or chemiluminescent.
  3. The intensity of the signal is directly proportional to the amount of captured antigen.
• While being highly specific and sensitive, the specificity of ELISA is lower compared to western blot.
• There are two main types of ELISA tests:
  • Direct ELISA: tests for the antigen directly
  • Indirect ELISA: tests for the antibody, which indicates the presence of an antigen (indirectly)
• A sandwich ELISA is an indirect ELISA that employs two antibodies that bind to different epitopes on the antigen of interest.

Direct ELISA

1. The patient's sample supposedly containing the protein of interest (i.e., the antigen) is added to a well of microtiter plates with a buffered solution.
2. The specific antibody-enzyme conjugate is added to the solution.
3. A substrate for the enzyme is added
4. Spectrometry is used to detect the generated chromophore
   • Higher concentration of antibodies binding to the antigen → stronger signal
   • Lower concentration of antibodies binding to the antigen → weaker signal

Indirect ELISA

• Same procedure as direct ELISA, with the following exceptions:
  • The antibody specific for the antigen of interest is not labeled itself and is called primary antibody.
  • The primary antibody is detected by a secondary, labeled antibody.

Sandwich ELISA

1. A surface plate is coated with capture antibodies (not the patient's antibodies).
2. The sample is added to the coated plate where the captured antibodies bind the antigen of interest.
3. Specific (labeled) antibodies for the antigen are added. ; if the antigen is present, the antibody binds to the antigen.
4. A substrate for the enzyme is added (color, fluorescent, or electrochemical changes are due to the reaction between substrates and enzymes).
5. Spectrometry, fluorescence, or electrochemical studies are performed to assess for the amount of antigens present.

Uses

• Screening for HIV antibodies (high sensitivity, low specificity)
• Testing for West Nile virus antibodies
• Detection of the following organisms:
  • Mycobacterium tuberculosis
  • Rotavirus (in feces)
  • Hepatitis B virus
  • E. coli enterotoxin (in feces)
• Detection of PSA

References:^[3]

Overview

• Polymerase chain reaction (PCR) is a technique that allows amplification of specific chromosome segments by producing more than a billion copies. This makes testing of very small sequences of DNA that could not otherwise be studied possible.
• Common sequences of DNA studied by PCR include mutations, microsatellite instability sequences, and short tandem repeats (STRs).

Procedure

A PCR usually consists of 25–50 cycles, which are divided into three phases.

1. Denaturation
   • The sample is heated; at a target temperature of 90–98°C (194–208°F) for 20–30 seconds.
   • High temperature breaks the hydrogen bonds between complementary base pairs; of the double-stranded DNA and produces two single DNA strands.
2. Hybridization
   • The sample is cooled; at a target temperature of 50–65°C (122–149°F) for 20–40 seconds.
   • The following is added to the sample:
     • Complementary DNA primers (a short sequence of DNA nucleotides on which DNA polymerase initiates replication) that are necessary for annealing
     • Enzymes (for DNA synthesis): heat-stable DNA polymerase (Taq DNA polymerase)
     • Deoxyribonucleotides (dNTPs)
   • Primers bind the 3′ ends of the DNA sequence that needs to be amplified.
3. Elongation and amplification
   • The sample is heated; at a target temperature of 70–80°C (158–176°F); 72°C (162°F) is most commonly used for DNA polymerase.
   • DNA polymerase uses dNTPs to elongate the primers, thereby replicating the sequence of the sample DNA strands.
   • The process is repeated until it yields an amplification to 10⁶ –10¹⁰ copies of the original DNA fragment (approximately 25–50 cycles).

Uses

• Detection of HIV (particularly when ELISA and western blot are inconclusive)
  • Useful for the detection of neonatal exposure to HIV
  • Positive early after infection
  • Not affected by the immune status of the patient (e.g., immunocompromised patients may not produce an antibody response)
• Diagnosis of bacterial and viral infections (e.g., Lyme disease, HSV encephalitis)
• Diagnosis of immunodeficiency (e.g., severe combined immunodeficiency)
• Forensic analysis (e.g., comparison of DNA samples from suspects, paternity testing)
• Sequencing of mutations

Reverse transcription polymerase chain reaction (RT-PCR)

• Procedure
  1. A sample mRNA is converted to complementary DNA (cDNA) by reverse transcriptase.
  2. cDNA is amplified by the standard PCR procedure (see above).
• Use: : detection and quantification of mRNA levels in a sample

Real-time polymerase chain reaction (also known as quantitative PCR, or qPCR)

• Procedure
  • A PCR technique that utilizes fluorescence (e.g., intercalating dyes or DNA probes) for monitoring amplification of targeted DNA during the PCR via computer software (in a graph).
  • Melting temperatures specific to the amplified fragment allow for high specificity.
  • There are many types, including quantitative reverse transcription PCR (RT-qPCR) and semiquantitative real-time PCR.
• Use
  • Allows monitoring of a targeted product at any point throughout the amplification
  • Rapidly detect nucleic acids for diagnosis (e.g., RT-qPCR for SARS-CoV2)
  • Easily quantify gene expression (e.g., via comparing to a standard curve of serial dilutions)
  • Quantify and genotype viruses

Molecular biological methods are used in the (prenatal) diagnosis of inherited disorders, e.g., to detect gene mutations. They are also used in the diagnosis of infectious diseases (e.g., diphtheria), in forensics, and in tumor diagnostics. Specimens include DNA from nucleated blood cells or, in prenatal diagnostics, chorionic villi.

Genetic markers

Numerous loci vary dramatically in a population. Repetitive sequences of various lengths are present in several noncoding regions of the genome. These repetitive sequences differ in sequence motif length. The frequency of repetition differs in each individual. Polymorphisms in DNA form the basis, e.g., for the diagnosis of diseases and allow individuals to be identified.

• Genetic markers (or DNA markers): individual differences in the DNA sequence of a specific region of the genome
• Three different markers are used for DNA analysis.
  • Microsatellites (short tandem repeats, STRs): repetitive sequences of several base pairs in DNA that are highly polymorphic
    • Analyzed via PCR to identify individuals Very small amounts of DNA suffice for analysis.
    • Belong to the VNTRs (variable number tandem repeats): Short nucleotide sequences in the genome of an individual that are repeated a variable number of times.
  • SNPs (single nucleotide polymorphism): DNA sequence variants in a population that differ by only a single base pair. They are usually caused by errors during DNA replication and are point mutations.
  • RFLP (restriction fragment length polymorphism): Depending on the DNA sequence of an individual, cleaving of chromosomal DNA with restriction enzymes leads to DNA fragments of variable lengths.
    • The fragments are analyzed using Southern blot and detected by probes.
• Genetic fingerprint: the DNA profile of an individual, especially as determined by microsatellite analysis

Detection of gene mutations (DNA diagnostics, molecular genetic testing)

DNA diagnostics is suitable for the direct or indirect detection of a gene mutation that results in disease. It can also be used to exclude the presence of a gene mutation.

Direct detection

Various methods can be used in the direct detection of gene mutations.

• Prerequisite: The causal gene for the (suspected) disease is known.
• Process
  • Amplification of a specific region of the affected gene using PCR
  • Detection of a mutation, e.g., through sequencing or cleavage of DNA fragments using restriction enzymes
    • Restriction enzymes (restriction endonucleases)
      • Definition: enzymes that cleave double-stranded DNA at an enzyme-specific nucleotide sequence
      • Most restriction sites are palindromes.
      • Cleavage of DNA using restriction enzymes results in sticky ends or blunt ends.
      • Origin of restriction enzymes: prokaryotic defense system against foreign DNA
  • Analysis via gel electrophoresis: Detection is based on the altered mobility of the DNA fragments of mutated and normal alleles in gel electrophoresis.

Indirect detection (genetic linkage analysis)

Indirect DNA diagnostics are performed if direct detection is not possible.

• Prerequisite: The disease occurs in several family members and the suspected locus is known.
• Principle: analysis of genetic markers associated with the mutated gene and comparison of the patient's genotype with that of unaffected family members
• Result: There is no direct detection of a gene mutation, but a probability of the presence of a certain mutation that causes disease (genetic risk score) can be calculated. The validity of indirect DNA diagnostics depends on the pattern of inheritance and the number of family members being investigated.

Identification of chromosomes

Karyotyping can be used to visualize chromosomes for examining chromosome numbers and for an overview of potential structural changes within a chromosome. Staining is used to visualize the special banding patterns that are characteristic for every chromosome.

• Karyotyping
  • Determines number, size, morphology, banding pattern, and arm-length ratio of chromosomes
  • Can be performed on samples from various tissues (e.g., amniotic fluid, bone marrow, placenta, blood).
  • Helpful for diagnosis of trisomies, monosomies, and sex chromosome disorders.
• Banding pattern: transverse bands of various widths and distribution, which can be induced depending on the preparation and staining technique
  • Preparation
    • The cell sample is cultivated and cell division is stimulated.
    • To obtain chromosomes during metaphase, the cells are arrested using the spindle inhibitor colchicine.
  • Banding techniques
    • Staining with quinacrine (fluorescent bands; not used routinely in diagnostics)
    • Giemsa banding (standard banding technique, results in dark G bands with transcriptionally inactive chromatin and bright, transcriptionally active R bands)
  • Analysis: assessment of an average 10–15 metaphase chromosome pairs in 1250x magnification
• Karyotype
  • Evaluation of a karyogram according to the number and the structure (morphology, length, arm-ratio, pattern of banding, size) of the chromosomes
  • The total number of chromosomes is stated first followed by the type of sex chromosome present (e.g., 46, XX = healthy, female karyotype).
  • Anomalies, if present, are mentioned last (e.g., 47, XY+21: male karyotype with trisomy 21).

Fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization is a method used for the staining of specific DNA sequences by a fluorescence-labeled DNA or RNA probe, e.g., to stain chromosomes in karyograms, in tumor diagnosis, or to map specific genes on chromosomes in metaphase.

• Procedure
  • Denaturation of DNA in the prepared chromosomes
  • Hybridization of the DNA with a single-stranded, fluorescence-labeled DNA probe that is complementary to a specific DNA sequence
  • Analysis of the chromosome set in fluorescence microscopy
    • A fluorescent signal indicates the site of the bound probe.
    • Microdeletion: the deleted region does not exhibit fluorescence, compared to the region present in the same locus of the other copy of the chromosome.
    • Translocation: fluorescence signal from one chromosome is present in a different chromosome
    • Duplication: extra copies of chromosomes that exhibit fluorescence in addition to the normal ones
• Result
  • The specific staining increases the resolution compared to the classical staining methods of karyograms.
  • Even minor chromosomal aberrations (e.g., microdeletions) can be identified.
  • FISH is also possible on chromosomes in interphase.
• Use: direct visualization of chromosomal anomalies at specific gene loci on a molecular level
  • Detection of microdeletions (2–3 × 10⁶ base pairs) such as in DiGeorge syndrome
  • Search for evidence of a Philadelphia chromosome translocation t(9;22)

DNA microarray (array comparative genome hybridization, CGH)

Mainly used to simultaneously examine expression levels of multiple genes or to genotype many regions at the same time.

• Procedure
  1. Preparation of the sample
     • A sample (m)RNA and control (m)RNA are converted to complementary DNA (cDNA) by reverse transcriptase.
     • cDNA is then labeled with a fluorescent dye, one color for the sample that is being tested, another color for the control (e.g., green for the control, red for the sample being tested).
     • mRNA is removed and samples are combined.
  2. Preparation of the chip
     • Thousands of genetic sequences of nucleic acid (DNA or RNA) probes are attached to a chip (e.g., glass, silicon).
     • Both patient and control DNA is applied to this chip and hybridizes to the probes on the chip.
     • The chip is mounted to a scanner that can detect complementary binding of probes and sample sequences.
     • Each region of the chip stands for a known genetic sequence.
     • The higher the expression of the gene in one sample, the more intense the fluorescence.
• Uses
  • Helpful for detection of copy number variations (CNVs), single nucleotide polymorphisms (SNPs) that are used for genotyping, forensic science, cancer mutations, genetic linkage analysis, and genetic testing.
  • Sequencing: determination of the exact sequence of base pairs of a gene; e.g., to demonstrate an unknown mutation on a disease-causing gene

References:^[4]

Overview

CRISPR gene editing is a technique in genetic engineering that employs the prokaryote CRISPR/Cas9 defense system directed against foreign gene sequences to modify the genomes of living organisms (e.g., adding or deleting genes in DNA sequences).

• CRISPR (clustered regularly interspaced short palindromic repeats)
  • A family of DNA sequences of prokaryotic origin
  • crRNA (CRISPR RNA): transcribed CRISPR sequence; binds to Cas9
• Cas9 (CRISPR-associated system 9)
  • Enzyme (endonuclease) that produces single or double-strand breaks at a specific nucleotide sequence guided by a site-specific RNA (targeted DNA double-strand break)
    • In prokaryotes, the guide RNA consists of crRNA and tracrRNA.
    • For laboratory use, a single guide RNA is specifically designed and synthesized.
  • The cas9 gene sequence is found adjacent to the CRISPR sequence.
• tracrRNA (transactivating crRNA): RNA sequence that is partially complementary to crRNA; also binds to Cas9 (needed for Cas9 maturation)

Adaptive prokaryotic immune response

1. Foreign DNA is incorporated into own DNA at the CRISPR locus (acquisition).
2. The CRISPR locus is transcribed together with foreign DNA and forms the primary transcript.
3. The primary transcript binds tracrRNA and is processed to form a crRNA-tracrRNA hybrid, which contains a foreign genetic sequence.
4. crRNA-tracrRNA hybrid forms a complex with Cas9.
5. Now foreign DNA that contains the sequence complementary to the one contained by the crRNA/tracrRNA/Cas9 complex can be recognized and cleaved.

CRISPR/Cas9 in gene editing

For gene editing purposes, tracrRNA and crRNA are combined into one molecule, the single synthetic guide RNA (sgRNA) that is complementary to the DNA sequence of interest (target DNA). There are three major variants of Cas9 used in CRISPR gene editing:

• Wild-type Cas9
  • Cleaves dsDNA
  • Induced dsDNA breaks can be repaired using the wild-type Cas9 via the following pathways:
    • Nonhomologous end joining (NHEJ) pathway: deletion of the targeted gene (gene knock-out) → accidental frameshift mutations
    • Homology-directed repair (HDR) pathway: insertion of donor DNA (gene knock-in) → mutation in the gene of interest
• Cas9D10A
  • Cleaves only one DNA strand (nickase activity)
  • More specific because it does not activate NHEJ but only the high-fidelity HDR pathway
• Nuclease-deficient Cas9
  • Mutations in nuclease domains prevent cleavage but not binding.
  • Can be used to activate or silence genes by creating fusion proteins of Cas9 with effector proteins

Potential applications of CRISPR/Cas9

Some of the potential applications of CRISP/Cas9 system (not currently used in clinical medicine) include:

• Immuno-oncology: using manipulated immune cells to fight cancer
• Curing genetic diseases by replacing the alleles of genes associated with disease phenotypes with unaffected variants (e.g., sickle cell disease)
• Eliminating virulence factors of pathogens

Definition

A screening test that detects and quantifies the types of hemoglobins present in a sample by separating them based on their electrical charge.

Applications

• Diagnostic work-up of hemolytic anemias
• Screening for or evaluation of hemoglobinopathies in high-risk individuals (e.g., positive family history)
• Evaluation of abnormal erythrocytes on peripheral blood smear

Procedure

• A sample of the patient's hemoglobin is obtained by hemolyzing a sample of blood using a hemolysate reagent.
• The hemoglobin sample is added to the gel electrophoresis buffer.
• An electric field is applied to the buffer that causes the different hemoglobin types to separate according to their electrical charge.
• A stain is applied to the gel to make the charged molecules visible.
• Hemoglobin is negatively charged at an alkaline pH and migrates on the gel towards the anode, forming a band.
• The degree of negative charge of the hemoglobin molecule determines the migration speed and distance from the cathode to the anode.
  • HbA migrates the fastest and therefore the greatest distance, followed by Hb F, Hb S, and Hb A2, and Hb C (A > F > S > A2 and C).
  • Hemoglobin C migrates the least because a missense mutation replaces the negatively charged glutamic acid with the positively charged lysine.
  • Hemoglobin S migrates less than HbA because of the replacement of the negatively charged glutamic acid with the neutral valine.

A FaSt Car can go far (A > F > S > C).

Expected findings

Please also see “Hemoglobin patterns” in thalassemia, sickle cell disease, and hemoglobin C disease.

• Normal (adult): wide HbA band (AA)
• Normal (fetal: ): HbA and widened HbF band (AF)
• Beta thalassemia minor (trait): narrowed HbA band and widened HbA₂ band
• Beta thalassemia major: no HbA band; widened HbA₂ and HbF bands
• Sickle cell trait: HbA and HbS bands (AS)
• Sickle cell anemia: no HbA band; wide HbS band (SS)
• HbC trait: HbA and HbC bands (AC)
• HbC disease: no HbA band; wide HbC band (CC)
• HbSC disease: no HbA band; HbS and HbC bands (SC)

RNA interference (RNAi)

• Definition: inhibition of gene expression via small, non-coding RNA molecules that bind target RNA (mostly mRNA)
• Examples: miRNAs, siRNAs (See “Nucleotides, DNA, and RNA” for more information)
• Functions
  • Inhibition of gene expression
  • Defense mechanism against foreign RNA molecules
• Mechanisms
  • Inhibition of translation
  • mRNA destabilization
    • Removal of poly(A) tail at the 3′OH-end of target mRNA (deadenylation)
    • Decapping
  • mRNA degradation

Cre-Lox system

• Used to induce recombination between specific DNA sites (e.g., to study the effect of gene insertions or deletions)
• Consists of Cre recombinase and LoxP sequences
  • Cre recombinase: an enzyme that catalyzes recombination between LoxP sequences
  • LoxP sequences: short DNA sequences within the cell genome that have sites for Cre recombinase binding

Molecular cloning ^[5]

• Definition: experimental production of recombinant DNA molecules within host organisms (e.g., bacterial hosts)
• Process
  1. Isolation of eukaryotic target mRNA
  2. Production of complementary DNA (cDNA) using reverse transcriptase
  3. Insertion of cDNA fragments into a cloning vector (e.g., bacterial plasmids carrying the antibiotic resistance genes)
  4. Transformation of the produced recombinant plasmid into bacteria
  5. Selection of bacteria that contains the plasmid (e.g., via antibiotic exposure when cloning antibiotic resistance genes)
• Result: synthesis of multiple copies of target cDNA (cloned DNA)
• Application: human protein production in bacteria (e.g., insulin, recombinant factor VIII, human growth hormone)