Biology SAC: CRISPR, PCR, Gel Electrophoresis, Recombination and Transformation PDF

***[Protection of viruses:]*** Cas1 and Cas 2 run along any DNA looking for sequence NGG, called a PAM. Cas1 and Cas2 cut out a piece of DNA 20 bases upstream of the PAM, called a protospacer. Cas1 and Cas2 put the protospacer into a region in the CRISPR array at the 5 prime en.d RNA polymerase...

***[Protection of viruses:]*** Cas1 and Cas 2 run along any DNA looking for sequence NGG, called a PAM. Cas1 and Cas2 cut out a piece of DNA 20 bases upstream of the PAM, called a protospacer. Cas1 and Cas2 put the protospacer into a region in the CRISPR array at the 5 prime en.d RNA polymerase will run along the CRISPR array to transcribe pre-CRISPR RNA, then tracer RNA sticks onto the repeat regions of the pre-CRISPR RNA. RNAase cuts in the repeat region and cuts through the tracer RNA in the repeat region leading to gRNA. The gRNA gets picked up by Cas9 forming a CRISPR-Cas complex. Cas9 uses gRNA to guide the Cas9 to the right place in D.NA. If a cell is again infected, Cas9 will start to look for a PAM. When Cas9 finds a PAM sequence it will unwind the DNA and compare the sequence of bases in its gRNA with bases upstream of the PA.M If the sequence is complementary it cuts the viral DNA 4 bases upstream of the PAM within the protospacer region. on When the viral DNA is cut, enzymes within the bacterium will naturally act to repair it. However, the repair mechanisms in a cell are prone to errors that can result in nucleotide additions, deletions, or insertions in the middle of the viral gene. This is advantageous in the case of bacteriophage infiltration because these mutations tend to render viral genes non-functional. If a mutation does not occur after the cut, the gRNA will find the gene again and repeat the whole process until the DNA repair mechanisms induce a mutation, inactivating the virus. ***[Gene editing: ]*** 1 Synthetic sgRNA is created in a lab with a complementary spacer to the target DNA that scientists wish to cut. 2 A Cas9 enzyme is obtained with an appropriate target PAM sequence. 3 Cas9 and sgRNA are added in a mixture and bind to create the CRISPR-Cas9 complex. 4 The sgRNA-Cas9 mixture is injected into a specific cell, such as a zygote. 5 The Cas9 finds the target PAM sequence and checks whether the sgRNA aligns with the DNA. 6 Cas9 cuts the selected sequence of DNA. 7 The DNA has a blunt end cut that the cell will attempt to repair. 8 When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site. Scientists may inject particular nucleotide sequences into the cell with the hope that it will ligate into the gap. ***[PCR:]*** **Purpose of PCR:** to amplify DNA **How Does PCR Work:** Identify DNA that wants to be amplified(region of interest). Identity 25 base sequence at 3 prime end of each strand bracketing region of interest. **Building Primers:** DNA Primers are made complementary to the 25 base sequences, created by DNA synthesiser. ***[PCR Machine:]*** **95 Degrees Celsius:** DNA is denatured and breaks hydrogen bonds between complementary nucleobases, creating two single-strands **50 Degrees Celsius:** Short primers quickly annel to the region of interest before the 2 strands of DNA can annel back together **72 Degrees Celsius:** Taq polymerase attaches to both primers and adds complementary base pairs to create a complementary DNA strand. This is Taq polymerase optimal temperature. Extends from 5 prime to 3 prime direction. This process is repeated 25-30 times to leave copies of just the region of interest. ***[Gel Electrophoresis:]*** ***[Preparation of gel:]*** Agarose is melted and put onto a tray that creates wells at the end of the gel As gel sets it creates a porous structure that allows DNA to mo.ve The gel tray gets placed in an electrophoresis chamber with a buffer.r Buffer allows the conduction of electricity via sa.lt ***[Loading of Gel:]*** The gel is put in an electrophoresis chamber and covered with a buffer. Connect negative electrode to the side where the DNA is(wells) and the positive one on the other side where the DNA will go. **Removing gel:** Need to stain gel to make DNA visible ***[The smaller DNA fragments are faster and move further away from the negative electrode.]*** ***[The bigger DNA fragments are bigger and are closer to the wells:]*** ***[Recombination and Transformation:]*** Recombination: Joins DNA together with different sources Transformation: Changing Bacertia by introducing a plasmid to it. ***[How recombinant Plasmids are made:]*** Endonuclease cuts DNA in the middle of DNA. They will find a restriction site and cut at that restriction site. Every restriction enzyme has a specific restriction site. 6 base palindrome. Needs sticky ends [Step 1: ] An endonuclease is chosen to cut upstream and downstream of a gene leading to sticky ends. [Step 2: Choosing Plasmid]: The chosen plasmid must have 2 genes that encode for observable traits. One of these genes must contain the same restriction site of the endonuclease being used. [Step 3: Using the restriction endonuclease:] Create cDNA to remove the intron in DNA as bacteria don\'t have introns. This is done by the enzyme reverse transcriptase which turns mRNA into DNA Cutting both plasmid and the human gene out of cDNA, complementary sticky ends will be created and will most likely join together. If joined DNA ligase will create a sugar-phosphate bond along the backbone of the DNA. Only some plasmids will take the human gene while others don\'t. [Step 4: Transformation:] Mix both recombinant and normal plasmids with bacteria, bacteria naturally take up plasmids. Bacteria that take up a plasmid are now transformed bacteria. [Step 5: identifying transformed bacteria:] Culture bacteria on an agar plate that contains the observable trait without the restriction site. Ones that don\'t produce that trait don\'t have a plasmid in them. With the bacteria left put bacteria in another agar plate with an environment that codes for the other observable gene with a restriction site. Bacteria that don\'t show for that trait have the recombinant plasmid. Take this bacteria and nurture it in a fresh agar plate. Bioethics: 3 approaches: Consequence Based: Driven by consideration for the consequences likely to result, the aim is to maximise positive outcomes and minimise negative effects. Do whatever must be done to achieve the greatest good for the greatest number of shareholders. Duty/Rule-based: driven by fundamental duty to act in a certain way. The aim is to follow a set of rules and responsibilities with less regard for the consequences. Virtues-based: driven by the character of the person instead of rules and consequences. Aim to emphasise the moral nature of the individual and provide guidance to the behaviour a morally good person would hope to achieve. [Ethical Concepts:] Integrity: Commitment to Knowledge, Encouraging individuals to act truthfully and honesty when finding results and presenting findings Justice: commitment to fairness, encourages consideration of different people\'s opinions and positions especially those directly affected or marginalised by course of action. Beneficence: commitment to maximising benefits, acting in a way that benefits others, promotes personal well-being and good of other persons. Non-maleficence: commitment to minimising as much harm as possible, encourages to act in a way to remove as much harm as possible. Respect, commitment to consideration, and considering the values of others, including personal welfare, beliefs and freedom. Prioritises the freedom of others to make their own decisions mRNA (messenger RNA): Function: Carries genetic information from DNA in the nucleus to the ribosomes in the cytoplasm. It acts as a blueprint for protein synthesis, providing the instructions for building proteins. rRNA (ribosomal RNA): Function: Makes up the structure of ribosomes, which are the sites where proteins are synthesized. It helps bind mRNA and tRNA together during protein synthesis and catalyzes the formation of peptide bonds between amino acids. tRNA (transfer RNA): Brings amino acids to the ribosome during protein synthesis. Role: It has an anticodon that matches the codons on mRNA, ensuring the correct amino acids are added to the growing protein chain. ***[the genetic code as a universal triplet code that is degenerate ]*** [Universal triplet code: ] The genetic code is made up of sequences of three nucleotides, called codons, on the mRNA. Each codon specifies a particular amino acid or a stop signal during protein synthesis. The genetic code is considered universal because the same set of codons is used to encode amino acids in almost all living organisms, from bacteria to humans. The code is degenerate because multiple codons can code for the same amino acid. This redundancy helps protect against mutations, as a change in one nucleotide might still result in the same amino acid being added to the protein. ***[amino acids as the monomers of a polypeptide chain and the resultant hierarchical levels of structure that give rise to a functional protein ]*** 1. - 2. - 3. - 4. -