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Topic 8 - ID - Coggle Diagram

- - - - Induced Pluripotent stem cells
        
        iPS cells can be produced from adult somatic cells using appropriate protein transcription factors to overcome some of the ethical issues with embryonic stem cells.
        To do this, the genes that were switched off to make the cell specialised must be switched back on. This is done using Transcriptional factors.
- - - - Restriction endonucleases
        
        Restriction endonucleases are enzymes that cut up DNA.
        Thes naturally occur in bacteria as a defence mechanism.
        There are many restriction enzymes that have an active site complementary in shape to a range of different DNA base sequences, described as recognition sequences, and therefore each enzyme cuts the DNA at a specific location.
        Some enzymes cut at the same location in the double strand and create a blunt end.
        Others cut to created staggered ends and exposed DNA bases.
        The exposed staggered ends are palindromic and referred to as 'Sticky ends' because they have the ability to join DNA with complementary base pairs.
        
        Fragment cloning to amplify sample
        
        In vivo cloning
        
        Promoter and Terminator regions
        
        Restriction endonuclease cut at recognition sites leaving sticky ends.
        The DNA fragments must be modified to ensure transcription of these genes can occur.
        
        Promoter region must be added - added at the start of the fragment. This is a sequence of DNA which is the binding site of RNA polymerase to enable transcription to occur.
        Terminator region must be added - added at the end of gene. It causes RNA polymerase to detach and stop transcription, son only one gene at a time is copied into mRNA.
        
        Insertion of DNA into vector
        
        The plasmid is cut open using restriction endonuclease.
        This creates the same sticky ends.
        Therefore, the DNA fragment sticky ends are complimentary to the sticky ends on the plasmid.
        The DNA fragment and cut plasmid are combined and the enzyme ligase sticks them together (annealment).
        Ligase catalyses the condensation reaction to form phosphodiester bonds between nucleotides.
        
        Transformation
        The vector (plasmid with recombinant DNA) needs to be inserted into the host cell, where the gene will be expressed to create the protein required.
        To do this, the cell membrane of the host cell must be more permeable.
        To increase the permeability, the host cells are mixed with calcium ions and heat shocked.
        This enables the vector to enter the host cell's cytoplasm.
        
        1 more item...
        
        In Vitro Cloning
        Fragments of DNA can be amplified in vitro via the polymerase reaction chain (PCR).
        
        Equipment list
        
        Thermocycler
        DNA fragment to be amplified
        DNA polymerase - taq polymerase
        primers
        DNA nucleotides.
        
        PCR method
        
        1 - The temperature is first increased to 95 degrees to break hydrogen bonds and split the DNA into single strands.
        2 - The temperature is then decreased to 55 degrees so that primers can attach (annealing).
        3 - The enzyme DNA polymerase then attaches complementary free nucleotides and makes a new strand to align next to each template (synthesis). The temperature is increased to 72 degrees for this stage, the optimum for taq DNA polymerase.
        4 - Process repeated to make as many clones as needed.
        
        Advantages of PCR
        1 - Automated - more efficient.
        2 - Rapid - 100 billion copies of DNA can be made within hours.
        3 - Doesn't require living cells - quicker and less complex techniques needed.
      - Gene machine
        
        DNA fragments can be created in a lab using a computerised machine.
        Scientists first examine the protein of interest to identify the amino acid sequence, and from that work out what the mRNA and DNA sequence would be.
        The DNA sequence is entered into the computer, which checks for biosafety and biosecurity that the DNA being created is safe and ethical to produce.
        The computer can created small sections of overlapping single strands of nucleotides that make up the gene, called oligonucleotides.
        The oligonucleotides can then be joined to create the DNA for the entire gene.
        PCR can be used to amplify the quantity and to make the double strand.
        This process is very quick, accurate and makes intron free DNA so it can be transcribed in prokaryotic cells.
      - Reverse transcription
        
        PROCESS:
        1 - This enzyme makes DNA copies from mRNA.
        2 - Naturally occurs in viruses, such as HIV.
        3 - A cell that naturally produces the protein of interest is selected.
        4 - These cells should have large amounts of mRNA for the protein.
        5 - The reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA sequence.
        6 - Single stranded DNA is made (cDNA).
        7 - To make this DNA fragment double stranded, the enzyme DNA polymerase is used.
        
        ADVANTAGES:
        
        The cDNA is intron free because it is based on the mRNA template.
  - - - Collection and extraction
        
        The smallest sample of DNA can be collected for genetic fingerprinting. This could be from blood, body cells or hair follicles.
        If the sample of DNA is small, then PCR is used to amplify the amount of DNA.
        
        Digestion
        
        Restriction endonucleases are added to cut the DNA into smaller fragments.
        Enzymes which cut close to the target VNTRs are added.
        
        Separation
        
        The DNA samples are loaded into small wells in agar gel. The gel is placed in a buffer liquid with electrical voltage applied.
        DNA is negatively charged, so the DNA samples move through the gel towards the positive end of the gel.
        The agar gel creates resistance for the moving DNA, and smaller pieces of DNA can move faster and further along the gel.
        An alkaline is then added to seperate the double strands of DNA.
        Known as GEL ELECTROPHORESIS
        
        Hybridisation
        DNA probes are short, single stranded pieces of DNA complementary in base sequence to the VNTRs.
        The probes are radioactively or fluorescently labelled.
        Different probes are mixed with the single stranded DNA VNTRs on the agar gel for them to bind (hybridse).
        
        Developlement
        
        The agar gel will shrink and crack as it dries, and therefore the VNTRs and DNA probes are transferred to a nylon sheet.
        The nylon sheet can then be exposed to x-rays to visualize the position of radioactive probes, or UV light if fluorescence probes were used.
        
        Analysis
        The position of the DNA bands are compared to identify genetic relationships.
      - Uses of genetic fingerprinting
        Method can be used in fields of:
        
        Forensic science to place suspects at crime scenes.
        
        For medical diagnosis.
        
        To ensure animal and plants are not closely related before being breed.