Ch 2 DNA manipulation techniques and application Flashcards
What enzyme is used to synthesise DNA?
identify and explain
DNA polymerase
- unzips and makes DNA single-stranded
What enzyme is used to cut DNA?
identify and explain
endonucleases (aka restriction enzymes)
- cut DNA at specific recognition sequences known as restriction sites -> splits DNA into smaller fragments
- blunt ends ends of DNA fragment with no overhanging bases after being cut
- sticky ends ends of DNA fragment with overhanging cut ends
What enzyme is involved in joining DNA?
DNA ligase
- catalyses joining of two double-stranded DNA fragments at sugar-phosphate backbones
- bonds are strong covalent bonds
What is CRISPR-Cas9?
name, definition, overall function
Cluster Regularly Interspaced Short Palindromic Repeats
- definition -> tool for precise and targeted genome editing that uses specific RNA sequences
- bacterial defence mechanism against viruses (adaptive response)
What are the steps in CRISPR-Cas9?
6 steps
- bacteriophage (virus) insets itself into bacterial cell since it is unable to copy its own DNA
- DNA endonucleases cut viral DNA and fragment is stored in CRISPR tool/library called spacer
- transcription of spacers occur and CRISPR RNA is produced and binds with tracer RNA to make gRNA complementary to spacers
- guide RNA (rRNA) binds to Cas9 proteins to make CRISPR-Cas9 complex and gRNA will guide around cell
- CRISPR scans bacterial cell for any bacteriophage DNA complementary
- another bacteriophage will insert itself but CRISPR will recognise DNA, scan the DNA until PAM sequence is identified, then cut
How can CRISPR-Cas9 be used?
- insert new section of DNA like a different allele
- knock out DNA segment to turn a gene off
- edit faulty allele in gene
- activate or repress gene
- treating genetic diseases through alterin mutation or allele
What is a Polymerase Chain Reaction (PCR)?
function and 4 steps
- used to amplify or make multiple segment of DNA accurately and quickly
1. denature keep DNA around 95.C for two minutes to separate double stranded DNA into single strands
2. anneal DNA is at 55.C for two minutes while primers (complementary to sequence needed) binds to denatured DNA by base pairing at either end of region
3. extension DNA is kept at 72.C for one minute while Taq polymerase extends DNA adding nucleotides until two complete strands are formed
4. repeat
What is Gel electrophoresis?
definition and function
definition -> technique for sorting mixture of DNA fragments through electric field on basis of different fragment lengths
- phosphate backbone of DNA/RNA is negatively charged
- electric field will cause DNA fragments to migrate to postively charged side
- result is series of parallel bands of DNA fragments of differing distances down gel
What are the step in separating fragments through Gel electrophoresis?
6 steps
- DNA sample with fragments of varying sizes is combined with DNA load dye
- mixture is placed in well at one end of agarose gel
- agarose gel is immersed in buffer solution (salt solution)
- gel is exposed to electric field with positive (+) pole [anode] at far end and * negative (-) pole [cathode]* at starting origin
- smaller fragments move through gel faster than larger fragments
- fragments appear as bands on gel that can be interpreted in various ways under UV light
What are recombinant plasmids?
definition
plasmids that carry foreign DNA
What are vectors?
agent used to transfer pathogens or genes between cells and organisms
What are plasmids?
small circular piece of double-stranded DNA that is able to reproduce independently and may be taken up by cells in addition to chromosomal DNA
How to make recombinant plasmids?
4 steps
- DNA of plasmids is cut using endonuclease in order to create sticky ends (changing from circular to linear)
- foreign DNA fragments are cut by same endonuclease to have sticky ends that are complementary
- foreign DNA fragments and plasmids are mixed – sometimes sticky ends pair by weak hydrogen bonds
- DNA ligase is added causing permanent joining through covalent bonding
What is bacterial transformation?
definition
process where bacterial cells take up segment of foreign DNA that become part of genetic make-up
How to get plasmids into bacterial cells?
2 main steps and their definition
-
electroporation technique uses brief exposure of host cell
- cells are briefly placed in electric field that shocks and create holes in plasma membrane to allow entry of plasmid -
heat shock technique to transform bacteria where cells are suspended in ice cold solution and moved into warm solution to increase plasma membrane fluidity
- bacterial culture is placed in ice bath and chilled
- recombinant plasmids with resistence allele are added to bacterial culture and chilled
- bacteria and plasmidmix and placed in hot water (42.C) for 50 seconds to produce heat shock
- mix is returned to ice bath for two minutes
- bacteria are plated and incubated overnight – those that have not taken up plasmids will die
