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bio 3/4

dna manipulation

QuestionAnswer
What is an endonuclease (restriction enzyme)? A type of enzyme that cuts DNA into smaller (more useable) fragments by cleaving the phosphodiester bond, helps isolate the region of interest.
What is a ligase? A type of enzyme that joins fragments or repairs breaks of DNA through ligation.
What are the two types of ligations? Sticky end leaves DNA fragment with overhang and ligation is specific, however blunt ends leaves a clean-cut end, where the ligation is random.
What is a polymerase? A type of enzyme that catalyse the formation of polymers in particular nucleic acids. For example, RNA polymerase assembles RNA, DNA polymerase assembles DNA and Taq polymerase is commonly used in PRC.
What is the purpose of using PCR? Increased amount of DNA sample, large enough to be used and analysed in other techniques.
What is PCR? Method to amplify specific target sequences of DNA. PCR mixture requires a DNA sample, Taq polymerase, 2 DNA primers, nucleotides and a buffer.
What is the first step in PCR? Denaturation step. The sample is heated to 95 degrees, hydrogen bonds break, single strands of DNA are produced.
What is the second in PCR? Annealing step. Temperature is lowered to 50-60 degrees; primers anneal to complementary sequences on opposite strands.
What is the third step in PCR? Extension step. The sample is heated to 72 degrees, and Taq polymerase attaches to primers, move along adding free nucleotides forming a double stranded helix of target DNA.
What are some PCR applications? Gene cloning, vaccine production, drug discovery.
What is the purpose of gel electrophoresis? Separates fragments of negatively charged DNA based on length.
What are the steps of gel electrophoresis? Gel contains small well of DNA samples at the negative terminal of the gel chamber. A known DNA ladder is added for comparison. Buffer is added. DNA travels towards the positive end of the terminal.
How to interpret gel runs? Smaller fragments of DNA travel faster, therefore the fragments closer to the positive end of the chamber are smaller.
What is a plasmid? Small circular DNA molecules found in bacterial cells, often used as vectors to move target DNA.
What is a bacterial transformation? Genes inserted into plasmids which are incorporated into bacterial cells. Bacteria then self-replicate, expressing the proteins they encode.
What is a recombinant plasmid? Genetic recombination to bring together genetic material from multiple sources.
Why use a plasmid as a vector? Due to their small size, they are easy to manipulate, they have a range of restriction sites, suits many needs, and fast meditation.
What are the steps to create recombinant plasmid? Cut target DNA using sticky end endonucleases and the bacterial plasmid using the same endonucleases. Target DNA and plasmid are then permanently ligase together.
What is a transformed cell? A cell that contains foreign DNA.
What are the two methods to transform a bacterial cell? Heat shock involves placing the plasmid into an ice-cold solution, then rapidly increasing the temperature. This disrupts the plasma membrane. Electroporation involves an electric current sent to alter the plasma membrane.
What are some methods to know if the transformation is successful? Plasmids contain an additional ampicillin resistant gene. This allows transformed bacteria to survive on a dish of ampicillin. Another method is to incorporate a green fluorescent protein, successfully transformed bacterial will glow green on a dish.
What is reverse transcription? Converts mature mRNA into cDNA, then DNA polymerase will turn it into double stranded DNA helix
Created by: lucyokane
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