Methods of Investigation

Role of Methods in Genetic Engineering

In genetic engineering, DNA is the central “material” being examined, cut, copied, moved, and read. The methods in this chapter are a basic toolbox that allow biologists to:

• isolate and cut DNA in a targeted way
• join DNA pieces together
• separate and visualize DNA fragments
• detect whether particular sequences are present
• make many copies of selected DNA fragments
• determine the exact sequence of nucleotides
• introduce DNA into cells and organisms

Each of the following sections introduces one key method on a beginner level. Detailed molecular mechanisms and specialized variations are treated elsewhere.

General Steps in a Genetic Engineering Experiment

Most genetic engineering projects, regardless of the specific goal, follow a similar logical sequence:

1. Obtain DNA
• from cells, viruses, tissues, environmental samples, etc.
• often followed by purification to remove proteins, lipids, and other molecules.
2. Cut and Modify DNA
• use restriction enzymes to cut DNA at specific sequences
• sometimes modify ends (e.g., add short sequences, remove phosphates).
3. Combine DNA Fragments
• use ligases to join DNA pieces into new combinations
• often insert fragments into vectors (e.g., plasmids, viral genomes).
4. Introduce DNA Into Host Cells
• via various transfer techniques (chemical, electrical, viral, mechanical).
• cells take up the foreign DNA and may express it.
5. Select and Analyze Modified Cells
• check whether the desired modification is present, using methods like
• gel electrophoresis (fragment size)
• hybridization (sequence presence)
• PCR (amplifying specific sequences)
• DNA sequencing (exact nucleotide order).

These methods are often combined; for example, a cloning experiment might use restriction digestion, ligation, transformation (transfer into bacteria), PCR, gel electrophoresis, and sequencing in sequence.

Basic Concepts for DNA Handling

Before going into individual methods, a few practical concepts recur in almost every technique:

• Buffers: aqueous solutions with defined salt and pH conditions that keep enzymes active and DNA stable.
• Temperature control: many reactions require specific temperatures (e.g., 37 °C for many bacterial enzymes; cycling temperatures in PCR).
• Enzymes from microorganisms: a large part of the toolkit (restriction enzymes, polymerases) comes from bacteria, archaea, or their viruses.
• Contamination control: because DNA is everywhere and easy to transfer, sterile tools and clean procedures are essential.

With these basics in mind, we can now focus on individual investigation methods.

Restriction Enzymes and Ligases

Restriction enzymes and ligases are essential tools for cutting and joining DNA. As a pair, they enable the construction of recombinant DNA molecules.

Restriction Enzymes: Cutting DNA at Defined Sites

Restriction endonucleases (restriction enzymes) are bacterial enzymes that recognize short, specific DNA sequences and cut the DNA at or near these sites.

Recognition Sites

• Typical recognition sites are 4–8 base pairs long and often palindromic, meaning the sequence reads the same in the 5'→3' direction on both strands.
  Example:
• EcoRI recognizes 5'-GAATTC-3'.
• Because the sites are short, they occur many times in long DNA molecules, generating a characteristic pattern of fragments.

Types of DNA Ends Produced

Different restriction enzymes leave different types of ends:

• Sticky (cohesive) ends:
• The enzyme cuts the two strands offset, leaving short single-stranded overhangs.
• These overhangs can base-pair with complementary overhangs from DNA cut with the same enzyme.
• Blunt ends:
• The enzyme cuts both strands at the same position, leaving no overhangs.
• Blunt ends are less specific in their pairing but can still be joined by ligases.

Uses in Genetic Engineering

• Creating defined DNA fragments for:
• cloning into vectors
• mapping the structure of DNA molecules
• preparing DNA for further analysis (e.g., gel electrophoresis).
• Generating compatible ends so that fragments from different sources can be combined.

Because each restriction enzyme recognizes a specific sequence, choosing the right enzyme(s) is a crucial step in experimental design.

DNA Ligases: Joining DNA Fragments

DNA ligases are enzymes that create covalent bonds between DNA fragments, sealing breaks in the sugar–phosphate backbone.

Role in Cells and in the Lab

• In cells, ligases:
• repair DNA breaks
• join Okazaki fragments during DNA replication.
• In genetic engineering, ligases:
• connect DNA fragments with compatible ends
• complete the construction of recombinant DNA.

Ligation Reactions

A typical ligation reaction involves:

• DNA fragments with compatible ends (sticky or blunt)
• DNA ligase (often T4 DNA ligase in the lab)
• ATP or another energy donor (depending on the ligase)
• Appropriate buffer and temperature

Sticky ends greatly facilitate ligation because complementary overhangs base-pair first, bringing the ends into close alignment. Blunt-end ligation is less efficient and often requires higher DNA concentrations.

Combining Restriction Enzymes and Ligases

A common workflow:

1. Cut both the vector (e.g., plasmid) and the insert (DNA fragment of interest) with the same restriction enzyme(s).
2. Mix the cut vector and insert; their sticky ends base-pair.
3. Add ligase to seal the nicks in the backbone.
4. Introduce the ligated DNA into cells (transfer methods), then select for cells containing the recombinant DNA.

This combination is foundational for constructing genetically modified organisms and for many downstream analysis methods.

Gel Electrophoresis

Gel electrophoresis is a method to separate DNA (or RNA) fragments by size by applying an electric field to a gel.

Principle

• DNA molecules are negatively charged due to their phosphate backbone.
• When an electric field is applied:
• DNA migrates toward the positive electrode (anode).
• Smaller fragments move more easily through the gel matrix and travel faster than larger ones.

Setup

• Gel material: usually agarose for standard DNA separation.
• Wells: small depressions at one end where DNA samples mixed with a loading dye are placed.
• Buffer: conducts electricity and maintains suitable pH and salt conditions.
• Electric field: applied across the gel; voltage influences speed and resolution.

Visualization

After running the gel:

• The DNA is stained with a dye that binds to DNA and can be seen under UV or other specific light sources.
• The pattern of bands reveals the distribution of fragment sizes in the sample.

A DNA size marker (ladder) containing fragments of known lengths is run alongside the samples to estimate fragment sizes.

Applications

• Checking whether a restriction digestion has worked as expected.
• Estimating the size of DNA fragments (e.g., PCR products).
• Isolating specific fragments from the gel by cutting out bands and purifying DNA from the gel slice.
• Assessing DNA purity and integrity.

Gel electrophoresis provides a quick and relatively simple way to analyze DNA and is often used in combination with other methods.

Hybridization

Hybridization techniques are based on the property that complementary single-stranded nucleic acids can base-pair with each other.

Basic Idea

• Double-stranded DNA can be denatured (for example, by heating or chemical treatment), separating the two strands.
• When conditions are returned to normal, strands can renature (reanneal) with any complementary sequences present.
• If a labeled DNA or RNA fragment (called a probe) is added, it will bind specifically to sequences in the sample that are complementary to it.

Probes

Probes are:

• short or long pieces of DNA or RNA
• with a defined sequence
• labeled so they can be detected (e.g., radioactive labels, fluorescent tags, or enzymatic labels that produce color or light).

Hybridization Methods

Hybridization can be combined with several formats, for example:

• On filters (blots):
• DNA or RNA is separated by electrophoresis and then transferred to a membrane.
• A labeled probe is added, which binds to complementary sequences on the membrane.
• After washing away unbound probe, bound probe is detected, revealing which bands contain the sequence of interest.
• In situ hybridization:
• Probes hybridize to DNA or RNA inside fixed cells or tissues, showing where specific sequences or transcripts are located.

(Details of specific blot types and in situ hybridization are typically covered in more advanced chapters.)

Applications

• Detecting whether a given DNA sample contains a specific gene or sequence.
• Measuring RNA expression (which RNAs are present, and in what tissues or developmental stages).
• Mapping gene locations on chromosomes or within genomes.

Hybridization is powerful because it leverages the fundamental specificity of base pairing to answer “yes/no” questions about the presence and position of target sequences.

Polymerase Chain Reaction (PCR)

PCR is a method to amplify a defined DNA segment in vitro, generating millions of copies from minute starting amounts.

Key Components

• Template DNA: contains the region to be amplified.
• Primers: two short synthetic DNA oligonucleotides (usually 18–30 nucleotides) that flank the target region and define its boundaries.
• DNA polymerase: a heat-stable enzyme (commonly Taq polymerase) that synthesizes new DNA strands.
• dNTPs: the building blocks (A, T, G, C).
• Buffer and Mg²⁺ ions: conditions needed for enzyme activity.

Temperature Cycles

PCR proceeds in repeated cycles of three main steps:

1. Denaturation (~95 °C)
• Double-stranded DNA melts into single strands.
2. Annealing (~50–65 °C)
• Primers bind (anneal) to their complementary sequences on the single-stranded templates.
3. Extension (~72 °C for Taq polymerase)
• DNA polymerase extends the primers, synthesizing new complementary strands.

After each cycle, the target DNA region is doubled (in ideal conditions), leading to an exponential increase in copies.

Variants (Overview Only)

• Reverse transcription PCR (RT-PCR): starts from RNA, which is first converted into DNA by a reverse transcriptase.
• Quantitative PCR (qPCR): allows measurement of how much DNA (or corresponding RNA) was in the starting sample.

(These advanced uses and their interpretation are treated elsewhere.)

Applications

• Detecting the presence of specific genes or pathogens (e.g., in diagnostics).
• Amplifying DNA fragments for cloning or sequencing.
• Genotyping (identifying alleles or variants).
• Forensic analysis (very small amounts of DNA, e.g., from traces).

PCR is incredibly sensitive but also susceptible to contamination, so careful technique is crucial.

DNA Sequencing

DNA sequencing determines the exact order of nucleotides (A, T, G, C) in a DNA molecule.

Classical (Sanger) Sequencing (Conceptual Overview)

The classical Sanger method uses:

• A DNA template and a primer.
• DNA polymerase.
• Normal nucleotides (dNTPs).
• Special chain-terminating nucleotides (ddNTPs) that stop synthesis when incorporated.

When many synthesis reactions are performed in parallel with randomly incorporated ddNTPs:

• A mixture of DNA fragments of different lengths is produced.
• Each fragment ends where a particular base was incorporated.
• Separating these fragments (e.g., by electrophoresis) and detecting which ddNTP is at the end of each fragment reveals the sequence.

Modern instruments automate this process, using fluorescent labeling and capillary electrophoresis.

Next-Generation Sequencing (NGS) (Brief Introduction)

Newer methods can sequence millions of DNA fragments in parallel:

• The DNA is fragmented and each fragment is sequenced simultaneously.
• Large amounts of sequence data are generated quickly and at lower cost per base.

(Details of different NGS platforms and their data analysis exceed the scope of this introductory chapter.)

Applications

• Determining the sequence of genes or entire genomes.
• Identifying mutations in individuals or populations.
• Studying biodiversity via environmental DNA.
• Supporting personalized medicine by analyzing patient-specific genetic variants.

Sequencing is the ultimate form of “reading” genetic information and is often the final step in characterizing genetic constructs or natural DNA samples.

Transfer of Foreign Genetic Material

Once DNA has been cut, modified, and possibly amplified, it often needs to be introduced into living cells for expression, replication, or further modification. Methods to achieve this are collectively called DNA transfer or transformation/transfection techniques.

General Challenges

• DNA is a large, negatively charged molecule that does not easily cross cell membranes.
• Cells may actively degrade foreign DNA.
• Conditions must be chosen so that DNA enters cells without killing them.

Different types of cells (bacteria, yeast, plant cells, animal cells) often require different methods.

Common Transfer Techniques (Overview)

Chemical Transformation (Especially in Bacteria)

• Cells are treated with certain salts (e.g., CaCl₂) that make their membranes more permeable to DNA.
• A brief heat shock facilitates the uptake of plasmid DNA.
• Used widely in bacterial cloning.

Electroporation

• Short electrical pulses create temporary pores in the membranes of cells.
• DNA in the surrounding solution can enter through these pores.
• Applicable to many cell types, including bacteria, yeast, plant, and animal cells.

Viral-Mediated Transfer (Transduction)

• Modified viruses are used as carriers (vectors) for foreign DNA.
• The virus infects the target cell, delivering its genome (which now contains the desired DNA).
• Widely used in research and some forms of gene therapy.

Microinjection and Particle Bombardment

• Microinjection: DNA is physically injected into cells using fine glass needles.
• Often used for large cells, such as animal eggs.
• Particle bombardment (gene gun): DNA is attached to tiny metal particles, which are shot into cells or tissues.
• Often employed in plant transformation.

(Each of these techniques has specific technical details, efficiency levels, and safety considerations that are treated in more specialized chapters.)

Selection and Verification

After DNA transfer, usually only a fraction of cells have taken up and correctly integrated or maintained the foreign DNA. Therefore:

• Selection markers (such as antibiotic resistance genes) are often used to identify cells that contain the foreign DNA.
• Verification methods (PCR, gel electrophoresis, hybridization, sequencing) confirm that the desired construct is present and intact.

Combining Methods: Typical Workflows

In real experiments, the methods in this chapter are rarely used in isolation. Examples of method combinations include:

• Cloning a gene:
1. Isolate DNA.
2. PCR amplify the gene.
3. Cut both PCR product and plasmid with restriction enzymes.
4. Ligate insert into plasmid.
5. Transform bacteria.
6. Select transformants.
7. Verify plasmids by restriction digest, gel electrophoresis, and sequencing.
• Detecting a pathogen:
1. Extract DNA from patient sample.
2. Use PCR with pathogen-specific primers.
3. Analyze products by gel electrophoresis (presence/size).
4. Optionally sequence to confirm identity.

Understanding what each method can do, and its limitations, is essential for designing robust genetic engineering experiments and for interpreting their results correctly.