Molecular techniques are methods used to analyze and manipulate DNA, RNA, and proteins to study biological processes.
• Blot: In molecular biology, blot is a technique for transferring DNA, RNA or proteins onto a carrier or membrane so they can be separated by use of gel electrophoresis.
• Probe: A short single stranded DNA which is complementary to desired DNA.
• Hybridization probe: A hybridization probe is a short (100-500bp) single stranded DNA radiolabeled that is complementary to desired DNA.
Southern Blotting
- Southern blotting is an example of RFLP (Restriction fragment length polymorphism).
- Developed by Edwin M. Southern (1975).
- Southern blotting is a hybridization technique for identification or detection of particular DNA sequence from other similar molecules.
Procedure Steps
1) DNA extraction: Extracting the genomic DNA/desired DNA from the organism.
2) Restriction digest: The DNA cut down into smaller fragments by using restriction enzymes.
3) Gel electrophoresis: The desired DNA fragments are separated by gel electrophoresis.
4) Denaturation: After electrophoresis, the SDS gel is soaked in alkali (NaOH) or acid (HCL) to denature the ds DNA fragment.
5) Blotting: The separated strands of DNA are then transferred to positively charged membrane-nylon nitrocellulose membranes by the process blotting.
6) Restriction Hybridization with labelled probe: The DNA bound to membrane is then treated with labelled probes which are complementary sequences to the gene of interest. The probe binds with the complementary DNA on the membrane.
7) Visualisation by Autoradiogram: Visualise the labelled DNA probe on membrane under autoradiogram which give patterned bands Indicating the presence of the target DNA sequence.
Capture an image of the membrane using X-ray film detection system.
Application of Southern Blotting
- Used to detect DNA in given sample.
- Used paternity, criminal identification victim identification
- To isolate and identify desire gene of interest
- Used in diagnosis of disease caused by genetic defects.
Northern Blotting :
A technique used in molecular biology to study gene expression by detection of RNA (or isolated mRNA) in sample.
Developed by James Alwine & George Stark (1979)
Key to this method is by hybridization: Forming a double stranded DNA-RNA hybrid molecule between a single stranded DNA probe & a single stranded target RNA.
Method/Steps/Process :
1) Preparation of RNA from Sample: All the target mRNAs molecules should be extracted from from the sample.
2) Separated by Gel electrophoresis: In gel electrophoresis, the mixture of mRNA molecules are separated according to their size, amont and charge. using an electric field.
3) Blotting: In the next step the mRNA from the gel electrophoresis gel will be transferred onto a nylon membrane by the process of blotting.
4) Hybridization: The RNA bound to membrane is treated with labelled DNA probe for hybridization.
5) Washing: The blot membrane is washed to remove unwanted probe and RNA.
6) Visualisation by Autoradiogram: Visualize the DNA-RNA hybrid under autoradiogram which give patterns of band.
Application of Northern Blotting:
- Detecting a specific mRNA is a sample.
- Starting gene expression pattern
- Detection or diagnosis of diseases.
- Construct of cDNA library.
- In the screening of recombinants by detecting the mRNA produced by transgene.
PCR (Polymerase Chain Reaction) :
Polymerase Chain Reaction (PCR) is a technique used in molecular biology to amplify a single or a few copies of a piece of DNA to millions of copies of the DNA sequence.
PCR was invented by Kary Mullis (American biochemist) in 1983 and won the Nobel prize in 1993.
It involves cycles of denaturation, annealing, and extension, utilizing a DNA polymerase enzyme, primers, and nucleotides.
PCR is widely used in various applications such as genetic testing, forensics, diagnosis of infectious diseases, and molecular biology research.
The enzyme commonly used in PCR is Taq polymerase, which is derived from the thermophilic bacterium Thermus aquaticus.
Taq polymerase is heat-stable, allowing it to withstand the high temperatures during the denaturation step of PCR without becoming denatured itself.
Steps of Polymerase Chain Reaction (PCR) :
1.) Denaturation: Heating the DNA sample at 91-95°C for 20-30 seconds to separate the double-stranded DNA helix into single strands.
2.) Annealing: This step is typically performed at a lower temperature, usually around 55-60°C for 20-40 seconds. This allows the primers to bind (anneal) to their complementary sequences on the single-stranded DNA template.
3.) Extension: DNA polymerase synthesizes new DNA strands by extending from the primers, using nucleotides in the reaction mixture at 72°C.
4.) Repeat: These steps are repeated for multiple cycles, typically around 20-40 times, to exponentially amplify the target DNA sequence.
Sanger DNA Sequencing:
Principle: The Sanger DNA sequencing developed by Fredrick Sanger in 1977 for determining the sequence of nucleotides in DNA molecules.
The process involves DNA replication in the presence of dNTPs and ddNTPs leading the production of DNA fragment of varying length, added on the gene of interest.
These fragments are then separated by size gel electrophoresis, revealing the sequence of identified the terminated positions. The result is a series of bands, representing the original DNA sequence.
Method/Steps/Process
1) Primer binding: The DNA sample to be sequenced by binding of primero at specific location ‘in template strand.
2) DNA synthesis: DNA polymerases deoxyribonucleotide (dNTPs) and dideoxyribonucleotides (ddNTPs) are added to for the synthesis of DNA. DNA polymerase synthesises a new DNA strand by using the dNTPs.
3) Chain Termination: Chain terminating ddNTPs are randomly incorporated during synthesis, leading to terminated fragments of varying length.
4) Gel electrophoresis / Fragment Separation: DNA fragments of different length are separated by gel electrophoresis creating sequencing ladder.
5) Sequence determination: The sequence is determined by identifying the fluorescent label terminating ddNTPs allowing the reconstruction of the original DNA sequence.
DNA Fingerprinting:
1) Sample Collection: Obtain DNA samples from the person using buccal swabs on blood samples.
2) Isolation of DNA: Extract DNA from the collected samples using specialised techniques to separate the genetic material from other cellular components.
3) PCR Amplification: Replicate multiple copies of specific regions of theDNA through PCR technique.
4) DNA fragmentation: The amplified DNA is fragmented by the restriction enzymes.
5) Gel Electrophoresis: Separate the DNA fragments based on size by gel electrophoresis.
6) Southern Blotting: Transfers the separated DNA fragments from the gel to a membrane.
7) Hybridization: Adding labelled DNA probes that bind specifically to the target DNA sequences, labelling them for detection.
8) Autoradiography: Visualise the labelled DNA fragments through autoradiography revealing a unique pattern of bands of person’s DNA.
9) Analysis : Analysis the DNA band patterns of that person. Probability of paternity based on the DNA pattern, considering the prevalence of biological relationships.