DNA Manipulation Flashcards
What is DNA amplification?
Why is it used?
- DNA amplification is the process of replicating an identical target DNA. Generating millions of copies in the process.
- DNA amplification is used by scientist if their is limited amounts of target DNA. A large trace of target DNA allows scientist to analyse using other techniques. E.g Can be used at crime scenes if there is limited amounts of DNA.
What is the Polymerase chain reaction?
Outline the process of PCR.
- PCR is the process used to amplify DNA. It replicates millions of copies of DNA within a few hours.
2.
- Denaturation: DNA is heated up to 90 degrees to break the DNA’s hydrogen bonds to single stranded DNA from double stranded DNA.
- Annealing: DNA is heated back to 50-60 degrees to allow the primers to attach to the DNA in opposite sides and opposite ends of the Single stranded DNA.
- Extension: DNA is heated back to 72 degrees to allow the Taq polymerase to attach to the DNA strand. It moves along the DNA adding new nucleotides to form 2 double stranded DNA.
Why is the DNA polymerase Taq polymerase used in PCR?
- Taq Polymerase is used in PCR as it has heat resistant properties making it effective in the process of PCR.
(BECAUSE PCR IS HEATED TO 92C, THEN 50-60 THEN 82)
What is Gel electrophoresis?
When is this used in DNA manipulation?
Why is it used?
Other information
- Gel electrophoresis is the process of separating DNA fragments according to their size.
2.
- Gel electrophoresis is used after the stages of PCR.
- Used by scientist to detect mutations by comparing individuals traits with another individual.
- Used to compare DNA left at the
crime scene to potential suspects.
4.
- Electric current is added to the gel, causing the DNA fragments to move from the negative terminal to the positive terminals.
- Largest fragment is located at the negative terminal whereas the smaller fragments are located at the positive terminal.
- Smaller fragments move faster than the larger fragments.
What are restriction enzymes (endonucleases)?
Two types of restriction enzymes? Explain what they are.
- Endonucleases is when DNA is cut into smaller useable fragments to isolate (seperate) particular gene of interest.
- Sticky-end restriction enzymes and blunt end
- Sticky end is the process in which the DNA backbone is cut at different location, exposing the bases. ^- —-_
- Blunt end is the process in which DNA is cut at the same location leaving clean cuts. |—|
What are ligases?
What are Ligation of sticky ends and blunt ends?
- Ligase is an enzyme that joins DNA and RNA together.
- Exposed ends first binds to its complementary base pair, ligase permanently joins the sugar phosphate backbone.
-Random. Any two fragments can join if into contact, ligase joins the sugar phosphate backbone.
What is Recombinant DNA?
Recombinant DNA is when two different DNA from different organisms are joined together.
What are plasmids?
Why are they used as vectors (carriers)?
- Plasmid are small circular DNA molecules found in bacterial cells.
- They are small, easy to manipulate.
- Carry a range of restriction sites.
- Fast replicating when out into a host bacterial cell.
Outline the process of creating a recombinant DNA.
- Target DNA is cut out using a sticky end cut and then isolated (separated).
- Plasmid is cut out with the same cut as the Target DNA, sticky end cut. Plasmid and Target DNA now both have exposed ends that are complementary to each other.
- Target DNA and plasmid and are placed together.
- Ligase is added to rejoin the sugar phosphate backbone of the DNA.
What is reversed transcriptase?
Edd
Explain the selection and screening of transformed bacteria stage.
Bacteria that have incorporated a foreign plasmid will have be able to survive as it is resistant to a particular antibiotic.
- When exposed to a certain substance, those without the the recombinant DNA will be killed off.
