Molecular Biology Techniques: Hybridization, Sequencing, and PCR

Northern Blotting Techniques

• Definition and Purpose: Northern blotting is a technique closely related to Southern blotting but specifically designed for the detection of RNA rather than DNA.     

  • It identifies specific RNA sequences using complementary probes.     

  • The most frequent application is determining gene expression levels, specifically whether a gene is being transcribed into mRNA within a particular sample or tissue.

• Key Principles of Gene Expression:     

  • Gene expression is spatio-temporally regulated; not all genes are expressed in every tissue or at all times.    

  • A single gene may produce multiple mRNA transcripts of varying sizes.

• The Northern Blotting Workflow:    

1. RNA Extraction: Single-stranded RNA is extracted from the sample.

2. Electrophoresis: The extracted RNAs are applied to an agarose gel to be separated.     

3. Transfer (Blotting): The separated RNA molecules are transferred (blot) onto a nitrocellulose membrane.     

4. Hybridization: hybridise with ssDNA probe that is complementary to the gene of interest.    

5. Detection: wash and detect probe to show if the RNA of the gene of interest was present (and estimate its size)

• Applications of Northern Blotting:     

  • Determining tissue-specific gene expression.     

  • Studying the regulation of gene expression levels over time, such as during different stages of biological development.

In Situ Hybridization (ISH)

• Methodological Overview:     

  • In situ hybridization is a technique used to detect specific RNA molecules directly within cells or tissues, without blotting.     

  • Probes (either DNA or RNA) hybridize to the target mRNA at its functional site within the cell (in situ).    

  • The procedure can be performed on thin tissue sections or on whole organisms.

• Significance in Developmental Genetics:     

  • The technique is widely used to determine "where and when" genes are expressed.     

  • It reveals the precise cellular location and tissue distribution of specific mRNAs when viewed under a microscope.     

  • It can also be utilized to locate specific genes or sequences on whole chromosomes.

• Labeling and Detection Methods: Colorimetric In Situ Hybridization (CISH):         

  • Uses a color-generating affinity probe.         

  • Example: Digoxigenin-labeled probes detected by an antibody conjugated to a colorimetric enzyme, such as Alkaline Phosphatase.         

  • Case Study: Detection of myogenin mRNA in mouse embryos. $Myogenin$ is essential for muscle development.     

• Fluorescent In Situ Hybridization (FISH):         

  • Uses fluorescently labeled probes for visualization.         

  • Tissue Localization Example: Simultaneous use of five different probes in a Drosophila embryo, each labeled with a distinct fluorescent molecule.         

  • Revealing chromosome location of genes/sequences, example: Identifying deletions in the Duchenne muscular dystrophy (DMD) gene. In a heterozygous female, the DMD gene probe appears red while the X chromosome centromeres appear green. A deleted gene is indicated by an X chromosome lacking the red signal.

Summary of Gene Expression Hybridization Techniques

• Northern blots separate and detect RNA (gene products) using DNA probes.

• These techniques reveal the timing (developmental stage) and location (tissue type) of gene expression.

• DNA probes used in high-resolution microscopy on "chromosome spreads" can detect gene locations, mutations, or chromosomal count abnormalities (ploidy).

DNA Sequencing Principles and Sanger Method

• Definition and Utility:     

  • DNA sequencing provides a base-by-base sequence of genes within a genome.     

  • Sequence data allows for the prediction of amino acid sequences and the properties of encoded proteins.     

  • It reveals genome organization, including regulatory elements like promoters and enhancers, as well as structural features like telomeres and centromeres.

• The Sanger Method (Dideoxy/Chain-Termination Sequencing):

  • Developed by Fred Sanger, who was awarded the Nobel Prize in Chemistry in $1980$.     

  • Core Mechanism: Uses DNA polymerase to copy a single-stranded DNA template.     

  • incorporation of special dideoxy-nucleotides (ddNTPs) to terminate DNA strand extension - dideoxy sequencing

  • can be automated, enabling rapid sequencing of large numbers of DNA frags.

• Standard Nucleotides (dNTPs): DNA is typically replicated using four deoxynucleotides: dATP, dCTP, dGTP, dTTP. These form phosphodiester bonds between the 5’ -PO4 group of the incoming nucleotide and the 3’ -OH group of the previous nucleotide.     

  

  • Dideoxynucleotides (ddNTPs): These special nucleotides lack a hydroxyl -OH group on the 3’ carbon. Once a ddNTP is incorporated, the DNA polymerase cannot add further nucleotides because there is no 3’ OH to form the next phosphodiester bond, resulting in chain termination.

• Ideoxynucleotide sequencing:    

  • 4 reactions are needed to sequence the entire DNA fragment - one for each nucleotide A,C,G,T

  • dsDNA is seperated (denatured) into single strands

  • all dNTPs plus a small amt of each of the 4 ddNTPs are fluorescently labelled with a diff colour and are added together with DNA polymerase and primer

  • in each reation the primer is extended to copu the DNA frag, the rxn will terminate at diff points if a ddNTP is incorporated

  • rxn are repeated in many cycles until a fragment of every possible length is made

• Automated DNA Sequencing:     

  • The resulting fragments are separated by capillary gel electrophoresis.     

  • each ddNTP is fluorescently labelled with a diff colour determined by nucleotide at the end of it that leads to termination

  • As fragments pass a laser, their fluorescent color (representing the terminal nucleotide) is recorded.     

  • The sequence of recorded colors reveals the sequence of the DNA.  

  • increased throughput compared the the Sanger method

Genomic Sequencing and Evolution of Technology

• Whole-Genome Shotgun Sequencing:     

  • Sanger sequencing typically reads fragments of 30-1000bp

  • In the shotgun approach, the entire genome is broken into small fragments, each fragment is sequences and the complete genome is reconstructed by computationally assembling overlapping sequences

    

• Next-Generation Sequencing (NGS):     

  • Also known as massively parallel sequencing.     

  • Sequences millions of short fragments in parallel vy the sequential addition of nucleotides to DNA fragments on a solid surfae   

  • enables rapid, high-throuput genome cale analysis

• sequencing technologies

  • original sequencing too 10 years and $2 billion, now possible to sequence human genome for less than $1000

  • third generation sequencing allows faster, cheaper sequencing and longer reads

  • companies exist to provide personal genome sequences

• The Human Genome Projects:     

  • reference genome, built from a small number of individuals

• one thousand genomes project

  • sequenced 1092 human genomes from across the globe

  • Catalogues the basis of genetic variation in humans to explain individual differences in susceptibility to disease, response to drugs or reaction to environmental factors

• humans are 99.9% identical

• single base pair differences between individuals can explain differences in disease risk, drug response

• Genomic sequence in other organisms

  • genomes of more than 340 eukaryotes have been sequenced

  • + 30000 bacteria

Polymerase Chain Reaction (PCR)

• Fundamental Concept:     

  • Invented by Kary Mullis (Nobel Prize $1993$).         

  • A method for selectively amplifying a specific DNA sequence.  

  • The Enzyme: Uses Taq polymerase, a thermostable enzyme derived from the thermophilic bacterium Thermus aquaticus, to copy DNA. This enzyme functions at high temperatures.

  • rapidly amplifies specific DNA fragments without the need for cloning vectors or restriction enzymes

  • requires primers that bind to known DNA sequences that flank the target region

  • extremely sensitive, can amplify DNA from pico to femtogram quantities

  • widely used in diagnostic and forensic science

• The PCR Cycle (Three Steps):     

• uses repeated cycles of heating and cooling to copy a specific DNA fragment

1. Denaturation ($\sim 95^\circ C$): temperature is increased for ard 30s to seperate DNA strands into single strands.    

2. Primer Annealing ($\sim 45$-$68^\circ C$): Temp is decreased for ard 30s to allow primers to anneal to base pair to complementary sequences in target DNA template     

3. Extension ($72^\circ C$): Taq polymerase extends primer and synthesize nascent DNA strand for ard 1min.

4. PCR cycle 2: bi increasing reaction temp, the process begins again

• Amplification Dynamics:     

  • Amplification is exponential ($2^n$), doubling the number of copies every cycle ($2 \rightarrow 4 \rightarrow 8 \rightarrow 16 \dots$).     

  • Newly made DNA strands become templates in the next cycle

  • After $30$ cycles, one fragment can become approximately 1bil copies ($2^{30} \approx 10^9$).

  • amplified DNA frags can be separated by gel electrophoresis

  • PCR product size should be equal to the amount of DNA between primers

• Advantages and Clinical Utility:     

  • makes millions of DNA copies without cloning

  • Sensitivity: Can amplify DNA from picogram ($10^{-12}\,g$) or femtogram ($10^{-15}\,g$) quantities.     

  • Applications:         

  • Diagnostic testing for bacteria, viruses, and genetic mutations.  

  • Forensic identification from trace samples (e.g., a single hair).

  • Food testing for pathogens (e.g., $E.\,coli$) or species contamination (horse or whale DNA).  

  • Paternity testing.

• Limitations:     

  • Requires prior knowledge of the DNA sequence to design appropriate primers.

  • Extremely sensitive to contamination.  

  • Typically limited to fragments between $10\,bp$ and $50\,Kbp$.

Advanced PCR Adaptations and Genotyping

Genotyping with Short Tandem Repeats (STRs):     * Used to distinguish alleles by size using PCR followed by electrophoresis.     * Clinical Example: An STR marker on a chromosome $17$ near the $BRCA1$ gene can predict carrier status for hereditary breast cancer. Females with specific inherited alleles may have a $40$-$90\%$ lifetime risk of the disease.

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