Principals Underlying Basic Molecular Genetic Techniques

Key Technologies to Study and Analyze Genes

DNA Cloning

purify and amplify DNA fragments of interest
recombinant DNA technology
Recombinant DNA: DNA that has been created artificially by combining DNA from different sources

DNA fragments lack of capability of initiating replication
Replicon: Replication origin in a molecules
Human DNA fragments need a suitable replicon to replicate within a bacterial cell.
The result is the formation of a recombinant DNA.

Insertion of DNA Fragments into Cloning Vectors

Overview

Isolated human DNA is long and needs to be fragmented for cloning.
Cloning vectors can only accept relatively small DNA molecules.
Fragmentation by physical shearing is not sufficient as it produces long fragments with heterogeneous ends.
To facilitate ligation, small DNA fragments with uniform ends are required.

Tools Required

Restriction Enzymes: Bacterial enzymes that recognize and cleave specific DNA sequences.
DNA Ligases: Enzymes that join DNA fragments together.

Restriction Endonucleases

They recognize specific short sequence elements within double-stranded DNA.
Cleavage occurs on both strands within or close to these sequences.
Bacteria produce restriction endonucleases with different sequence specificities.

Restriction Sites

Often short palindromic sequences.
Read the same in the 5ʹ to 3ʹ direction on both DNA strands.
Cleavage may produce overhanging 5ʹ or 3ʹ ends (sticky ends) or blunt ends.

Cloning Vectors

DNA fragments with sticky or blunt ends can be inserted into vectors.
Vectors are engineered to contain unique restriction sites for specific enzymes.

Plasmid Vectors

Double stranded, circular DNA molecules
E. coli plasmids are widely used as cloning vectors
Components:
- Origin of replication: allows the plasmid to replicate in E. coli
- Selectable marker: gene that confers antibiotic resistance
- Multiple cloning site: region with multiple unique restriction sites for insertion of DNA fragments

DNA Library

Genomic library:
- DNA library: DNA molecules are cloned into a vector molecule.
- Genomic library: a collection of DNA fragments that represent the entire genome of an organism.
Complementary DNA (cDNA) library:
- cDNA library: represents the mRNA expressed in a cell type.
- only expressed genes are represented.

DNA Cloning Limitations

Laborious and time-consuming
Not suitable for parallel amplification of many DNA fragments from diferent sources

PCR (Polymerase Chain Reaction)

PCR is a technique that allows the amplification of a specific DNA fragment with gene-specific primers.

It is a cell-free method

Pros:

Fast (minimum 1 hour)
Parallel amplification of many DNA fragments from different sources
Highly sensitive (can detect a single DNA molecule)

The Polymerase Chain Reaction (PCR) is a technique used in molecular biology to amplify a segment of DNA. Below is the summary in key steps:

PCR Steps

Enzoklop, CC BY-SA 4.0 https://creativecommons.org/licenses/by-sa/4.0, via Wikimedia Commons

Preparation: Gather necessary components:
- DNA template with the target sequence.
- Heat-stable DNA polymerase enzyme.
- Two PCR primers for the start and end of the target sequence.
- Deoxynucleotide triphosphates (dNTPs), the building blocks for new DNA.
- Reaction buffer with Magnesium ions for enzyme activity.
- A thermal cycler for temperature control.
Denaturation: Heat the mixture to make the DNA single-stranded.
Annealing: Cool down so primers can bind to their complementary DNA sequences.
Extension: Raise temperature for DNA polymerase to synthesize new DNA strands.
Amplification: Repeat the cycle multiple times, doubling the DNA amount each cycle.
Finalization: End with billions of copies of the specific DNA segment.

This process allows for the DNA to be sufficiently amplified for further analysis.

PCR Cycle

Denaturation
Annealing
Extension

This Cycle is repeated 25-35 times

PCR Primers

Designing PCR Primers

Specificity:
- Primer sequence: Primers should only bind to the target sequence.ce:
- Length: 17-28bp optimal.
- Nature of the template:
  - Melting temperature (Tm):
    - Optimal Tm: 52-65°C
    - Influenced by GC content, length, and concentration
    - Anneling temperature: 5°C below Tm
    - Working Approximation: Tm = 4(G+C) + 2(A+T)
  - GC content: 45-55% optimal
Secondary structures and primers homology
- Avoid regions of self-homology (within the sequence)
Self-homology: the presence of complementary regions within a single nucleic acid molecule.
- Avoid inter-primer homology that could lead to primer dimers (between two primers)
inter-primer homology: the presence of complementary regions between two different primers.
Amplicon size
- Avoid large amplicon size
Repeats and Runs
- Long runs of single base repeats larger than 4bp should be avoided
- Long runs of di-nucleotide repeats should be avoided

Gene Expression Analysis Techniques

Endpoint of PCR

Measures the amount of DNA at the end of the reaction, rather than during each cycle. It is semi-quantitive which means that it can be used to compare the amount of DNA in different samples, but it cannot be used to determine the exact amount of DNA in a sample.

Easy and Cheap
Not very accurate

Angarose gel electrophoresis is used to separate DNA fragments by size. The DNA is stained with a dye that fluoresces under UV light. The DNA is visualized as bands on the gel.

Genome Research Limited | License: CC BY 4.0

Real-time PCR (qPCR)

Measures the amount of DNA during each cycle of the reaction. Exponecial phase of the reaction is the best time to collect and analyse data.

SYBR Green I and TaqMan assays

Cao, Yiqi & Luo, Yu-Feng et al. (2020). Digital PCR as an Emerging Tool for Monitoring of Microbial Biodegradation. Molecules. 25. 706. 10.3390/molecules25030706.

TaqMan Probe-Based

TaqMan probe: a short single-stranded DNA molecule that is complementary to the target sequence.

Hybridization assay

Hybridization: the process of forming a double-stranded nucleic acid molecule from two complementary single strands of DNA or RNA.

Why do we measure gene expression?

gene expression is identical across all cells
tissue-specific gene expression
disease-specific gene expression
gene expression changes over time

Methods for gene transcription profiling

Microarray
RNA-seq
RT-PCR

RT-PCR (Reverse Transcription PCR)

Reverse transcription: the process of synthesizing a DNA molecule from an RNA template.

Why?:

RNA is unstable and easily degraded
In order to have more RNA for analysis, we need to convert it to a more stable form (complementary DNA, cDNA)

Components:

cDNA: complementary DNA
DNA polymerase: enzyme that synthesizes DNA
Two primers: one for the start and one for the end of the target sequence
dNTPs: building blocks for new DNA
Reaction buffer with Magnesium ions for enzyme activity
Thermal cycler for temperature control

Negative control:

No reverse transcriptase
No template

Why examine protein expression?

RNA is not always translated into protein
Study correlation between RNA and protein expression
Examine post-translational modifications

Quantification of protein expression

Enzyme-linked immunosorbent assay (ELISA)
Western blotting
Immunohistochemistry (IHC)

Semi-quantitative methods: indirect measurement of expression
Quantitative methods: direct measurement of expression

Western blotting is a semi-quantitative method that uses antibodies to detect specific proteins in a sample.

Immunohistochemistry (IHC) is a semi-quantitative method that uses antibodies to detect specific proteins in a tissue section.

Immuno-staining: \

qualitive not quantitive \

Quantification of signal intensity and/or localisation