Unit 3 - Genetic engineering.ppvvxtm.pptx

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1. Genetic engineering Department of Biotechnology, School of Applied Sciences Dr. Yuvashree M

2. Unit 3: Techniques in Genetic Engineering 12hr DNA sequencing ( Dideoxy method, Maxam and Gilbert method); Polymerase Chain Reaction (PCR); Gel electrophoresis - PAGE; DNA hybridization (Southern blotting); Protein detection (Western blotting).

3. DNA sequencing ( Maxam and Gilbert method)

4. Mechanism of DMS with High Temperature & NaOH Step 1: Methylation by DMS DMS methylates the N7 position of guanine (G) and, to a lesser extent, adenine (A) . This methylation weakens the glycosidic bond , making the base prone to loss . Step 2: Depurination (High Temperature & NaOH ) High temperature (≥90°C) and NaOH (alkaline conditions) promote depurination , causing the loss of the methylated guanine or adenine from the DNA backbone . This results in an abasic (AP) site , where a purine is missing . Step 3: Strand Cleavage Under alkaline conditions, the phosphodiester backbone is unstable at AP sites . This causes strand cleavage , leading to DNA fragmentation at the guanine and adenine positions .

5. Advantages Purified DNA can be read directly Homopolymeric DNA runs are sequenced as efficiently as heterogeneous DNA sequences Can be used to analyze DNA protein interactions (i.e. footprinting ) The DNA is incubated with and without the DNA-binding protein (e.g., transcription factors, nucleases, polymerases). In the sample with the protein, it binds to its specific DNA recognition site . Can be used to analyze nucleic acid structure and epigenetic modifications to DNA Unmethylated C appears as bands in both lanes. Methylated C (5mC) appears as a band only in the no-salt lane. Disadvantages It requires extensive use of hazardous chemicals. It has a relatively complex set up / technical complexity. It is difficult to “scale up” and cannot be used to analyze more than 500 base pairs. The read length decreases from incomplete cleavage reactions. It is difficult to make Maxam -Gilbert sequencing based DNA kits.

6. Dideoxy method

7. Advantages of Sanger Sequencing Sanger sequencing is considered the gold standard method for many research and clinical applications .  It provides highly accurate results. This accuracy is particularly useful for validating sequences obtained through other methods, such as NGS technologies. Sanger sequencing technology is well-established with a straightforward process. This leads to reliable and reproducible outcomes. Sanger sequencing is suitable for small-scale projects or those including short DNA regions .  Sanger sequencing produces relatively long read lengths . Data analysis from Sanger sequencing does not require complex bioinformatics tools and expertise often necessary for NGS data interpretation.  The quality of the data obtained from Sanger sequencing is high with clear and easily interpretable chromatograms.

8. Limitations of Sanger Sequencing Sanger sequencing can only sequence short fragments of DNA. It has a limited throughput. It can sequence only one fragment at a time, making it slow and expensive for large genomes. Sanger sequencing is not suitable for long DNA sequences or large-scale sequencing projects due to its complexity and cost.  It involves manual steps compared to NGS workflows and is a time-consuming method . The preparation and handling time for Sanger sequencing can be longer. It has low sensitivity which makes it hard to detect rare mutations and study complex mixtures. Sanger sequencing requires relatively high-quality and pure DNA samples as degraded or contaminated DNA can lead to unreliable results.

9. Polymerase chain reaction  ( PCR ) Taq polymerase Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called   Taq  polymerase , after the heat-tolerant bacterium from which it was isolated ( T hermus   aq uaticus ). T . aquaticus  lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and is most active around  70 degree c(a temperature at which a human or  E. coli  DNA polymerase would be nonfunctional). This heat-stability makes Taq polymerase ideal for PCR. As we'll see, high temperature is used repeatedly in PCR to  denature  the template DNA, or separate its strands.

10. The key ingredients of a PCR reaction are  Taq  polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized. The basic steps are: Denaturation   (96 °C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step. Annealing   (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA. Extension   (72 °C): Raise the reaction temperatures so  Taq  polymerase extends the primers , synthesizing new strands of DNA.

11. Polyacrylamide gel polymerisation  Polyacrylamide, used mainly for SDS-PAGE, is a matrix formed from monomers of acrylamide and bis -acrylamide. It’s strengths are that is it chemically inert – so won’t interact with proteins as they pass through – and that it can easily and reproducibly be made with different pore sizes to produce gels with different separation properties. The polymerisation reaction, shown in the diagram below, is a vinyl addition catalysed by free radicals . The reaction is initiated by TEMED , which induces free radical formation from ammonium persulphate (APS) . The free radicals transfer electrons to the acrylamide/ bisacrylamide monomers , radicalizing them and causing them to react with each other to form the polyacrylamide chain. In the absence of bis -acrylamide, the acrylamide would polymerise into long strands, not a porous gel. But as the diagram shows, bis -acrylamide cross-links the acrylamide chains and this is what gives rise to the formation of the porous gel matrix. 

12. https://www.youtube.com/watch?v=VgAuZ6dBOfs&t=196s