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BIOLOGY CHAPTER 13

QuestionAnswer
Polymerases enzymes that catalyse the formation of nucleic acids
DNA Polymerase Acts to assemble DNA Enzymes uses each strand of the DNA double helix as a template for building the new DNA strands, following the complementary base pairing rules
Reverse transcriptas DNA polymerase that synthesises single-stranded DNA (known as copy DNA or cDNA) using single-stranded RNA as a template cDNA made from the mRNA already have the introns spliced out
RNA Polymerase Acts to assemble RNA RNA does not require not require primers to start the synthesis of RNA Add nucleotides until a ‘stop’ sequence is reached to create a single piece
DNA amplification – Polymerase Chain Reaction Makes many copies of a piece of DNA using the Polymerase Chain Reaction (PCR) technique. PCR is carried out in cycles using a DNA thermocycler which alters the temperature in pre-programmed steps
Polymerase Chain Reaction ingredients A sample of DNA A source of four nucleotides (A, T, C and G) to build new DNA strands A heat resistant DNA Polymerase such as Taq polymerase Single stranded DNA primers- specify the start and finish of the target DNA for replication
Polymerase Chain Reaction Method 1 Ingredients placed together in plastic tube in a DNA thermocycler, a heating block that is able to change temperature very quickly
Polymerase Chain Reaction Method 2 Denaturation - Heated to 95 degrees celcius- this breaks the hydrogen bonds and separates the strands of DNA
Polymerase Chain Reaction Method 3 Annealing- thermocycler cooled to 50-60 degrees Celsius to allow the primers to anneal (bind) to their complementary DNA sequence. One primer binds to each of the two template strands
Polymerase Chain Reaction Method 4 Extension- Temperature increased to 72- the optimal temperature for DNA (Taq) Polymerase enzyme. Taq polymerase moves along the template strand starting from the primer and begins to add nucleotides (3' → 5') to form double-stranded DNA
Polymerase Chain Reaction Method 5 The thermocycler then heats to 95 degrees and the next cycle of strand separation, binding of primers and DNA replication begins. Typically 35-45 cycles are used to amplify a DNA sample
Gel electrophoresis Technique used for separating fragments of nucleic acids (DNA and RNA). The technique is also used to separate proteins of different size, charge and shape
How DNA moves in gel electrophoresis DNA is slightly negatively charged DNA moves to positive end of the gel Smaller fragments move faster than longer ones- therefore move further in the given time
DNA ladder A DNA ladder containing DNA fragments of known length is also run on the gel for comparison with the samples. This allows the length of the sample DNA fragments to be estimated.
Gel electrophoresis steps DNA test sample can be cut into fragments with restriction enzymes DNA and sample is placed in a well in a gel Gel is placed in an electrophoresis bath where it is covered in a controlled pH solution and powered by electric voltage
Restriction enzymes Recognise short sequences of bases in a DNA molecule called the recognition site They act as molecular scissors
staggered cut in DNA leaves DNA fragments with overhanging ends called ‘sticky ends’. The exposed bases are then able to form complementary base pairs through hydrogen bonding with nucleotides of other DNA molecules that have complementary sticky ends
Non-staggered ends created by cutting the sugar-phosphate backbone on both strands of the DNA molecule at the same location within the recognition site. Are more difficult to use in DNA manipulation processes that require the joining of specific fragments
Ligase Group of enzymes that join fragments of DNA or RNA in a process called ligation Role of DNA ligase in a cell is to join segments of newly replicated DNA and to repair breaks in DNA molecules. DNA ligase is required to stabilise the recombined molecule
Vector A vector refers to an agent (such as a plasmid or virus) that carries a passenger DNA into a cell
Plasmid A plasmid is a small circular DNA molecule found in many bacterial cells. Plasmids replicate independently of the chromosome.
Recombinant DNA When DNA from two different sources (from different organisms) is joined together, the resulting molecule is called recombinant DNA
Joining of the passenger DNA and plasmid (recombinant DNA steps) 1 1. The DNA of the plasmids is cut at one point using a restriction enzyme creating sticky ends
Joining of the passenger DNA and plasmid (recombinant DNA steps) 2 2. The DNA passenger fragments are prepared using the same restriction enzyme. The plasmid and target DNA now have the same sticky ends with exposed bases that are complementary to each other
Joining of the passenger DNA and plasmid (recombinant DNA steps) 3 The passenger DNA fragments and the plasmids are mixed, and their sticky ends pair
Joining of the passenger DNA and plasmid (recombinant DNA steps) 4 DNA ligase makes the joins permanent by rejoining the sugar- phosphate backbone of the DNA
Joining of the passenger DNA and plasmid (recombinant DNA steps) 5 Plasmids that are contain the DNA passenger are then selected and reintroduced into bacterial cells
Transformation Incorporating foreign DNA (for example a foreign plasmid) into a cell
Artificial bacterial transformation - heat shocking Involves placing bacterial cells and a mixture of recombinant and non-recombinant plasmids in an ice cold solution hen rapidly increasing the temperature to disrupt the plasma membrane allowing the plasmid to penetrate the membrane and enter the bacteria
Artificial bacterial transformation - electroporation Bacterial cells and a mixture of recombinant and non-recombinant plasmids are subjected to an electrical current that alters the plasma membrane. Again the plasmids are then able to enter the bacteria.
Selection and screening of transformed bacteria Plasmid vectors contain other genes, including a gene for antibiotic resistance. Incubated cells at 37 ̊C so that can reproduce and from colonies on agar plates that contain the antibiotic are bacteria cells that have survived and are therefore chosen
Created by: emmawalton05