Northern Hybridization Technique

Northern Hybridization

• Technique related to Southern hybridization.
• It identifies RNA molecules with specific sequences.
• Used to identify specific gene expression via transcribed mRNA in a tissue or sample.
• Not all genes are expressed at all times.
• Some genes are only transcribed in certain tissues or at certain times.
• Some genes produce multiple mRNAs of differing sizes.

Northern Hybridization Blot

• mRNA extracted from tissue/sample separated by size on agarose gel.
• mRNA transferred directly to membrane, no denaturing needed due to single-strandedness.
• Labeled DNA probe from specific gene hybridizes to RNA on membrane.
• Single-stranded DNA probe hybridizes to complementary mRNA molecules.
• Used to determine gene expression in a specific tissue by detecting mRNA transcript complementary to the probe.
• Determines if gene expression levels are regulated over time (e.g., during development).
• Determines mRNA transcript size and whether different sizes are made at different times or in tissues.

In Situ Hybridization

• Hybridizes a probe directly to RNA without blotting.
• Can be carried out in tissue sections or entire organisms.
• Probe is hybridized to the mRNA transcript in situ where the transcript is made.
• Useful in developmental genetics to identify where and when genes are transcribed in embryos.
• Aids in identifying cellular and tissue distribution of mRNAs from specific genes.
• Digoxygenin or fluorescently labeled probes are used.

In Situ Hybridisation Examples

• Mouse: In situ hybridization of myogenin in a mouse embryo, where myogenin is required for muscle development.
• Digoxygenin-labeled probes visualized using an antibody that recognizes digoxygenin, followed by a colorimetric reaction.
• Drosophila: In situ hybridization of five probes simultaneously in a Drosophila embryo, with each probe labeled with a different fluorescent molecule.

DNA Sequencing

• Allows full characterization of genes within the genome by sequencing DNA clones.
• Provides base sequence to predict the amino acid sequence in the encoded protein.
• Facilitates determination of some properties of the gene product.
• DNA sequence provides information about genome organization, including regulatory regions, satellite DNA, telomeres, and centromeres.

Sanger Sequencing Method

• Common method using DNA polymerase to copy single-stranded DNA.
• Employs 'dideoxy' nucleotides to interrupt DNA polymerase's copying ability.
• DNA polymerase cannot extend the nucleotide chain after incorporating a dideoxynucleotide.
• Dideoxynucleotide chain termination sequencing is automatable, allowing rapid sequencing of large amounts of DNA.

Deoxynucleotides

• DNA is normally replicated using deoxynucleotides (dNTPs).
• DNA polymerase incorporates nucleotides by forming a phosphodiester bond between the 5' PO4 group of the nucleotide being incorporated and the 3' OH on the previous nucleotide.

Dideoxynucleotides

• DNA sequencing uses dideoxynucleotides (ddNTPs), lacking a -OH on the 3' carbon.
• DNA polymerase cannot incorporate further nucleotides after a ddNTP due to the absence of a 3' OH to form a phosphodiester bond.

Dideoxynucleotide Sequencing

• When DNA is replicated by DNA polymerase with dCTP and ddCTP, some fragments end prematurely.
• The length of fragments depends on when ddCTP is incorporated.
• ddNTP causes chain termination.

DNA Fragment Sequencing

• Four parallel sequencing reactions are performed, one each for A, C, G, and T.
• Each reaction contains many copies of the DNA fragment to be sequenced.
• Double-stranded DNA is separated into single strands with a short primer that binds to one end of the sequence.
• All dNTPs and a small amount of either ddATP, ddCTP, ddGTP, or ddTTP are added with DNA polymerase and a radioactive tracer, 35S.
• The DNA fragment is copied until a ddNTP is incorporated, stopping the reaction at different points.

DNA Sequencing Gel

• Products from each sequencing reaction are run in separate lanes on a polyacrylamide gel.
• Polyacrylamide gel separates DNA fragments by length to a single nucleotide.
• Each radioactively labeled DNA fragment position is visualized by exposing the gel to X-ray film.
• The reactions are run side by side on a gel.
• Shortest bands (DNA products closest to the primer) travel fastest; the gel is read from the bottom up, all four lanes together.

Automated DNA Sequencing

• Automated DNA sequencers use a single reaction for each DNA sequence, including all four ddNTPs.
• Each ddNTP is labeled with a unique fluorescent marker.
• You can identify which ddNTP was added at fragment end by identifying its fluorescent color.
• DNA sequence fragments are separated on a capillary gel.
• The fluorescent label on each ddNTP has a different wavelength.
• As fragments move down the gel, they pass a laser.
• Laser light excites the fluorescent tag on each fragment as it passes.
• The wavelength of the fluorescence shows sequence of the DNA
• Wavelength is read as the fragment passes, recorded for analysis.

Sequencing Technologies

• Sequencing technologies advances allow for faster sequencing of larger amounts of DNA.
• Original radioactive dideoxy sequencing on gels averages 200-400bp per reaction and takes days.
• Fluorescent capillary dideoxy sequencing averages 800-1000bp, takes hours, and generates 10-20,000bp per hour.
• Next-generation sequencing (NGS) uses sequential nucleotide addition on microchips, short reads of 50-200bp, can sequence thousands to millions of DNA fragments, generating over 200-600Mbp per day.
• The original human genome sequencing (3200 Mbp) took 10 years, hundreds of investigators, and $2 billion.
• It is now possible to sequence a human genome in 2-3 days for $10,000.
• It will soon be possible to sequence a human genome for $1,000.
• Third-generation sequencing aims for faster, cheaper sequencing and a $500 genome.
• Companies such as 23andMe offer personal genome services.

Human Genome Sequence

• Advances allow to rapidly sequence genomes.
• Original human genome was sequenced from samples from a number of individuals.
• 1000 Genomes Project sequenced 1092 human genomes worldwide.
• It is used to investigate human variation and understand individual differences in susceptibility to disease, response to drugs, and environmental reactions.
• Human genomes are 99.9% identical, allowing identification of single base pair differences (SNPs).
• Human evolution and disease maps can be studied by SNPs.

Genomic Sequences

• Wide varieties of genomes have been sequenced.
• Drosophila the first multicellular eukaryote sequenced.
• Main model organisms sequenced include E.coli, Saccharomyces cerevisiae, C.elegans, mouse.
• Genome sequences are available at public database, Ensembl.
• 80 vertebrate genomes (Alpaca to Zebrafish).
• 65 other metazoan
• 43 plants
• 589 fungi
• Over 30,000 bacteria

Polymerase Chain Reaction (PCR)

• PCR is powerful and allows selective amplification of specific DNA regions.
• Kary Mullis received the Nobel Prize in 1993 for PCR invention.
• Requires knowledge of genome sequence to design primers.
• It uses thermostable DNA polymerase (Taq) to replicate the DNA region.
• Taq is isolated from bacteria that colonize hot thermal springs.
• PCR is an alternative method to make many copies cloning without cloning vectors or restriction enzymes.
• Sensitive, and can amplify DNA fragments from very small DNA amounts (1 picogramme, or even 1 femtogramme).
• Many applications, e.g. in forensics and diagnostics.
• Can amplify DNA to identify individuals or food contamination.

PCR Process

• Involves repeated cycles of targeted DNA replication to amplify DNA.
• Three steps - denaturation, primer annealing, and extension, repeated using a thermocycler to amplify DNA exponentially.
• The number of copies doubles in each cycle: 2, 4, 8, 16, 32, 64, 128, 256, etc.
• New strands and old strands serve as templates.
• After 30 cycles, about 2 to the power of 30 copies are generated, approximately 1 billion copies.

Steps of PCR

• Denaturation: Heat the reaction to 95°C to denature DNA into single strands.
• Primer annealing: Reduce temperature to 45-68°C to allow primers to hybridize to target DNA
• Primer extension: Raise temperature to 72°C to allow Taq polymerase to synthesize DNA.

PCR Reactions

• PCR reactions performed in a Thermocycler.
• Set machine to repeat through PCR steps:
  • 96°C, 30 seconds (denaturation)
  • 58°C, 30 seconds (annealing)
  • 72°C, 1 minute (extension)
  • Repeat 30 times
• Amplified DNA fragments are separated by gel electrophoresis.
• PCR product size equals the amount of DNA between the primers.

Limitations of PCR

• A limitation: some nucleotide sequence information must be known to synthesize primers.
• Minor contamination of sample DNA can cause issues.
• Cannot amplify long segments of DNA, only relatively short ones.

PCR Uses

• Amplifies DNA fragments from tens of bases to 5kb.
• Has a wide variety of uses.
• Can amplify whole genes or gene parts (from genomic DNA or cDNA) for cloning and molecular analysis.
• Can amplify specific regions of DNA from various samples.
• Can amplify DNA from limited sources like blood spots or fossils.
• Can identify victims after natural disasters or major accidents (e.g., World Trade Center).

Applications of PCR

• PCR routinely screens mutations to detect genetic disorders.
• PCR detects bacteria and viruses in humans and pathogenic bacteria, e.g. E. coli, in contaminated food.
• PCR techniques are used to analyze samples from single cells.
• PCR is used in forensics to identify individuals from body fluids and in paternity testing.
• It is used to check and confirm DNA constructs or transgenic animals.
• PCR enforces worldwide bans on illegal sale of certain animal products, e.g., whale meat.
• Reverse transcription PCR (RT-PCR) is used to study gene expression by examining mRNA production by cells or tissues.
• Quantitative real-time PCR (qPCR) allows researchers to quantify amplification.