Manipulating Genomes Flashcards
PCR
- DENATURATION
95°C
- DNA fragments mixed with DNA nucleotides and primers
- this temp breaks the H bonds between complementary base pairs
- The DNA strands separate - ANNEALING
55°C
- primers bind to complementary bases
- required for replication of the DNA strands - ELONGATION
72°C
- DNA polymerase adds bases to the primer, building up complementary strands of DNA and so producing double stranded DNA identical to the original sequence
Gel Electrophoresis
- DNA is amplified using PCR
- DNA is cut into smaller fragments using restriction enzymes
- DNA fragments are placed into the wells at the end of the agarose plate near the cathode (negative)
- The plate is immersed in a buffer solution which helps maintain the pH
- An electrical current is passed through the plate and DNA starts to separate as the negative phosphate group of DNA is attracted to the positive anode
- The shorter DNA fragments move further than the longer fragments
- Ethidium bromide is used as a stain to see the fragments
Purpose of DNA profiling
DNA is used to identify individuals and species
- paternity tests
- identify new species
- forensics
Southern blotting
- alkaline solution is added to the gel after electrophoresis and a nylon filter / nitrocellulose paper is placed over it
- DNA fragments ‘blot onto the filter’
- the alkaline solution denatures the DNA fragments so the strands separate and the base sequences are exposed
- a single stranded gene probe that has a radioactive label can then be applied to the filter and it will bind to the DNA in a highly specific pattern
DNA profiling steps
- DNA is prepared
- DNA cut with restriction enzymes and loaded into the gel
- Gel electrophoresis
- Southern blotting
Genetic engineering steps (basic)
- Isolation of desired DNA fragment (using restriction enzymes or reverse transcriptase)
- Multiplication of DNA fragment (PCR)
- Transfer into organism using vector
- Identification of cells with new DNA fragment
- Growth/cloning
Restriction endonucleases
Used to cut genes at specific base sequences (recognition sites)
DNA ligase
Used to join together the cut ends of DNA by forming phosphodiester bonds
Reverse transcriptase
Used to build double stranded DNA from single stranded RNA
- Isolation
Use reverse transcriptase
- mRNA acts as a template on which a single stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase
- double stranded DNA is formed in the template of the cDNA using DNA polymerase
- copy of DNA coding for original protein e.g. insulin
Use restriction endonucleases
- these cut up DNA
- some of these leave fragments with blunt ends, others with sticky ends
PCR primers are used to amplify the gene
- Insertion
- Insert the DNA fragment into a vector e.g. bacterial plasmid or weakened virus
-restriction enzyme cuts DNA at specific recognition site - annealing: DNA ligase joins DNA backbone back together
- Transformation
The transfer of DNA into suitable host cells can be done by:
- Heat shock treatment- bacteria alternated between 0°C and 42°C with calcium chloride, membranes become more porous
- Electroporation-high voltage pulse applied to cell to disrupt membrane
- Electrofusion- electrical fields help introduce DNA into cells
- Transfection- DNA packaged into a bacteriophage, which can then transfect the whole cell
- Recombinant plasmids- T1 plasmids are inserted into the bacteria which infects some plants
- Identification
- identify the host cells that have successfully taken up the gene by use of gene markers
- the colonies are shown under UV light and any cells that don’t glow have successfully taken up the plasmid because the fluorescent gene has been disrupted
OR
if antibiotic resistance gene was used then any cells that die have successfully taken up the gene
- Growth/cloning
Grow the cells from the colony that have the correct gene inserted in them
Benefits of genetic engineering
- use for research e.g. diabetes, cancer
- get more vitamins and minerals into food such as rice / improve food health
- plants better adapted for environment
- vaccines
- improve crop yield- increase growth, pest resistance
fewer pesticides used- better for environment and water
