Unit 3 AOS 1 Flashcards
Difference between DNA and RNA
- DNA is double stranded whilst RNA is single stranded.
- DNA has thymine whilst RNA has uracil.
- DNA also has deoxyribose sugar and RNA has ribose sugar
What are polymerase
Enzymes that catalyse the formation of polymers
What is the role of DNA polymerases
Unwinds the DNA double helix as a template to build the new DNA strands
Steps of DNA replication
- DNA is unwinded by the Helicase enzyme.
- Primer binds to the DNA template strand
- DNA polymerase attaches and moves along the strand adding bases from 5’ to 3’ ( left to right)
What is endonucleases
Also known as restriction enzymes. Range of enzymes responsible for cutting DNA
Restriction Enzymes
Target recognition site ( 4-6 bases )
How do restriction enzymes cut?
Cleave the phosphodiester bond of the sugar-phosphate backbone holding the DNA nucleotides together
Restriction Enzymes are also
A large group of enzymes that occur naturally in bacteria. They are a part of the bacteria’s cell defense system and target foreign DNA that may enter the cells such as DNA bacteriophages
Blunt End restriction enzymes
These guys leave clean cuts- cutting both sugar-phosphate backbone on BOTH STRANDS
Sticky end restriction enzymes
Cut DNA backbone at different places, leaving exposed bases for nucleotides
LIGASES
Are a group of enzymes that join fragments of DNA or RNA through ligation. This is the reverse of restriction enzyme’s roles
DNA ligase
Joins fragments of DNA. Join segments of newly replicated DNA and repairs breaks in DNA molecules. Also joins fragments from different organisms as long as they are cut with the same restriction enzyme
RNA ligase
Joins fragments of RNA
Ligation of Blunt ENDs
Is random, DNA ligase can join any 2 fragments together. Ligation blunt ends are more difficult to us in DNA manipulation processes that require the joining of specific frags
Ligation of sticky ends
Is SPECIFIC since the exposed bases are bound by complementary base pairing. Joins fragments by creating a phosphodiester bond between the 3’ OH and the 5’ phosphate end of the adjoining nucleotides. Also requires the fragments to be cut by the same restriction enzyme
PCR ( polymerase chain reaction)
Creates large quantities of identical DNA samples
What is used in PCR?
Targeted DNA to be amplified. Taq polymerase. Free nucleotides. Two DNA Primers
Why Taq polymerase used?
Because Taq polymerase is heat resistant and resists the changes in temp during PCR
What are Primers?
Single stranded RNA usually 30 bases long that are complementary to each end of DNA. Specifies start and finish of DNA frag that needs to be amplified
Steps in PCR?
- Denaturing: DNA sample heated to 95 degrees to break bonds b/w base pairs
- Annealing: Temp reduced to 50-60 degrees. Primers bind
- Extension: Temp increased to 72 degrees, Taq attaches each primer on the DNA strand and moves along each strand adding free nucleotides to form double stranded DNA.
DNA hybridisation
Measures the relatedness and its process includes: desaturation, hybridisation and melting
Gel Electrophoresis
Separates fragments of nucleic acids (DNA and RNA)
What charge is DNA?
DNA is negatively charged because of its phosphate Group
What gel is used?
Agarose gel
What are the terminals?
Where the loading wells are it is negative going to positive:
Neg -> pos
Large fragments and small fragments.
The smallest fragments travel the furthest and closest to the positive terminal. The larger fragments don’t travel very fast and are closer to to negative terminal
Genotyping
Genotyping uses gel electrophoresis to determine the genotype of an organism at particular allele
DNA profiling
Distinguishes between individuals on the basis of variable regions of their DNA
What are the variable regions?
They are non coding regions of DNA:
- Short tandem repeats (STR) ( 2-6 bases)
- Variable Number tandem repeats ( VNTR) ( 20-60 bases long)
Why aren’t coding regions used?
Coding regions are not useful as they code for the products/ functions that are the same for everyone
DNA profiling Steps
- DNA is extracted from the cells
- DNA of the 13 different VNTR regions is amplified by PCR
- DNA is cut with specific restriction enzymes that have recognition sites at either side of VNTR regions
- DNA fragments are loading into the wells and separated by gel electrophoresis( smaller alleles have fewer repeats and therefore migrate faster and further than larger alleles with more repeats
Heterozygous
Means having 2 different alleles of a particular gene or genes
Homozygous
Means having 2 identical alleles of a particular gene or genes
If the individual is heterozygous ?
For a VNTR is will appear as two bands in the gel
If the individual is homozygous ?
For a VNTR it will appear as one thick band in the gel
Genetic screening
Form of DNA profiling used to analyse an individual’s DNA for a particular version of a gene ( allele) they might be carrying
What is the Universal Nature of DNA
Means that the DNA of one species can be combined with the DNA of different species using DNA manipulation techniques
Genetic Engineering
Ability to remove a single or group ( in some circumstances) of genes from an organism and transfer them to another organism. The new organism is then able get the transferred gene protein synthesises to produce a fully functioning protein
Vectors
Vectors is a vehicles that carries DNA between its donor and recipient cells. Most common vector is a plasmid
Plasmids
Prokaryotic Cells: DNA forms double stranded circular chromosomes . Bacterial cells also contain small circular pieces of double stranded DNA called plasmids. Chromosomal and plasmid DNA are located in cytosol
Recombinant plasmid
DNA from a host organism combined with DNA from a donor organism
Why are plasmids useful vectors?
They are small so they can be easily replicated. They carry a range of restriction enzyme sites. They self replicate once they are placed inside a bacterial cell and at a faster rate than their chromosomal DNA
4 important features of Plasmid vectors
- Restriction enzymes sites ( Sites that can be reignited and cut so that the gene of interest can be inserted)
- Antibiotic resistance gene
- Reporter gene: A gene that creates an easily identifiable phenotype
- Origin of replication (ORI): A sequence that signals the start site for DNA replication in bacteria
Gene cloning
PRODUCTION OF EXACT COPIES of gene using various DNA manipulation techniques
What is the use of cloned genes
Treat disease by mass producing specific proteins
Gene cloning purpose
Purpose is to clone a particular gene and then produce large quantities of the protein expressed by the cloned gene
Gene cloning steps part 1
- Isolation the genes of Interest :( is the DNA coding for the desired protein product) and is also cut using a specific restriction enzyme
Gene cloning part 2
- Preparing the gene of interest: Eukaryotic genes undergo RNA processing whilst prokaryotic genes do not.
- mRNA becomes double stranded DNA though an enzyme ( Reverse transcriptase)- It first transcribes the single stranded RNA into single stranded DNA. Then DNA polymer mass uses the single stranded DNA as a template to synthesis a double stranded DNA. Once synthesised, PCR is used to amplify this gene sequence.
Gene Cloning part 3
- Opening up the plasmid: Plasmid is cut with the specific restriction enzyme. Making the initially circular DNA temporarily linear
gene cloning part 4
- Creating recombinant plasmids: The plasmid and gene of interest are mixed together with the enzyme DNA ligase so that the sugar-phosphate backbones of each join which results in a closed circular piece of DNA again.
This is called the recombinant plasmid, as it has ‘recombined’ with a piece of foreign DNA.
At this point, it is common for the circular plasmid to re-join itself with the help of DNA ligase, without taking up the gene of interest.
This is referred to as a non-recombinant plasmid.
Gene cloning part 5
- Transforming bacteria; Recombinant plasmids (and non-recombinant plasmids) are mixed with bacteria. They are then soaked in calcium solution and heat shocked. Opening up the pores increases the chance of them taking on the recombinant plasmid
Gene cloning part 6
- Bacteria reproduce: Through binary fission, resulting in clones forming that are genetically identical to the parent cell
Gene cloning part 7
- Identifying transformed bacteria: To identify which bacteria have taken up a plasmid, antibiotic resistance genes found naturally in plasmids are used.
Bacteria that have taken up the plasmid vector are called transformed bacteria, and vice versa for untransformed bacteria
- Transformed cells have foreign DNA in them
Gene cloning part 8
- Identifying transformed bacteria with the recombinant plasmid: The transformed bacteria who survive exposure to an antibiotic are now screened to determine which have taken up a recombinant plasmid, and therefore have the capacity to synthesis the protein coded for by the target gene
Gene cloning part 9
- Extraction and purification of the protein: The transformed bacteria transcribe and translate the plasmid which contains the gene of interest.
The insulin protein is extracted and purified, ready to provide to diabetic patients.
Difference between DNA polymerase and RNA polymerase
DNA polymerase is used in replication and RNA polymerase is used in transcription
Genome
Is a complete set of genes within the DNA of an organism
Proteome
Is the complete set of proteins expressed of an individual cell at any given time
Denaturation
The tertiary structure’s bonds of a protein are broken and the protein shape is altered. Resulting in the protein being biologically inactive.
Renaturation
When a protein that is partially denatured maybe able to fold again when the conditions are correct
What do we do when tryptophan levels are low ?
Increase transcription to raise the levels. RNA polymerase will bind to the promoter, transcribing copies of structural genes which will then be translated to relevant proteins
What do we do when tryptophan levels are high?
Stop transcription by the trp binding to the repressor ( changing its shape) which then allows it to bind to the operator. Repressor prevents RNA polymerase from binding to the promoter.
Attenuation
Prevents the completion of transcription to deal with high levels of tryptophan
Steps:
1. Leader Section ( b/w operator and trp E) encodes for a short polypeptide with an attenuator sequence. Attenuator sequence gets transcribed into mRNA becoming self complementary sections that can form various hairpin structure.
1. As transcription and translation are continuous processes in Prokaryotic cells, after the RNA polymerase starts transcribing the operon, a ribosome can attach to the forming transcript and being translating the leader region
Attenuation- Higher trp levels
Ribosome will translate quickly causing it to fall off after translating the leader peptide, allowing the terminator hairpin and an associated hair pin to form, making RNA polymerase detach —> ending transcription .
Attenuation- lower trp levels
The ribosomes will be stalled as it waits for the tRNA to find 2 trp a.a back to back. The slowness causes the formation of antiterminator ( non terminating hair pin) who prevents the terminator from forming allowing transcription to continue
Proteins Secretory Pathway
All proteins who are made in the ribosome and attach to the surface of rough endoplasmic reticulum end up being exported to organelles or outside the cell. Once the protein is made, RER will then send the protein inside a vesicle to the Golgi apparatus and then outside the cell via exocytosis
Protein formation > RER> Inside Vesicle> Golgi Apparatus> Exocytosis
Enzymes
Are proteins that speed up reactions within an organism by lowering activation energy
Advantages of Biochemical Pathway
- Able to regulate themselves ( saves time and energy and resources) as the cell is producing molecules that it needs
Anabolic Reactions
Combines substrates into a larger product. Products have more energy than reactants
Endergonic Reaction
Energy is absorbed ( A for anabolic and absorbed)
P>R
Graphs looks like an A
Catabolic Reactions
Combine substrates into smaller products
Exergonic Reaction
Energy is released
R>P
Graphs looks less of an A
Competitive Inhibition
Binds to the active site temporarily. Rate of reaction is restored through increasing the substrate concentration
Non- Competitive Inhibition
Binds to the allosteric site. Active site changes shape . Enzyme activity decrease
Cloning Human Insulin?
> Insulin-> quaternary protein structure composing of 2 polypeptide chains ( A and B chain). They are each synthesised in a separate bacteria!
The plasmid vectors used to clone the insulin genes also contain a structural gene marker called B-gal
The A and B chain gene are inserted immediately after the B-gal sequence
Lock and Key Model
> When a specific substrate fits to a specific enzyme and binds through the active site.
Where the enzyme will not function unless the correct complementary substrate binds perfectly to the active site>
Induced Fit Model
> Initially, the substrate is not a perfect fit for enzyme but upon the binding of the substrate, the active site of the enzyme is able to alter its shape to mould around the substrate
Coenzyme
> An organic molecule that contains carbon and bind to enzyme to help them function.
They can activate the enzyme by interacting and altering the active site shape or act as electron carriers, taking electrons to the active site and transferring them to the substrate
They are continuously recycled
Oxidation and Reduction of Cellular Respiration and Photosynthesis
Cellular Respiration: Oxidation of glucose. Energy releasing (exergonic)
Photosynthesis: Reduction reactions. Energy requiring (endergonic)
