Genetic Engineering Flashcards
(32 cards)
What way can genetic material be modified?
Insert foreign gene from 1 organism to another= transgenic
Altering an existing gene so that it’s product is changed
Changing a gene expression so that it’s translated more often or not al all.
What is genetic engineering?
Altering the genes in a living organism to produce a genetically modified organism with a new genotype.
What are restriction enzymes?
They cleave the DNA apart.
2 types
1. Endonuclease- cleave within DNA
2. Exonuclease -cleave at end of DNA
How are the 4 types of endonuclease classified?
Subunit composition
Cleaveage position
Sequence specify
Co factor requirement
What is the main endonuclease used in gene cloning?
Type 2
How do we isolate the gene?
Using restriction enzymes.
Cut at specific sites
Some cut straight across - blunt ends
Some make a staggered cut- sticky ends
What happens to the sticky ends?
These sticky ends anneal to another piece of DNA by comp base pairing but only if they have been cut by the same restriction enzyme.
What is PCR?
Polyemerase chain reaction. Used to amplify a number of copies of a specific region of DNA. Produce a large enough sample to be analysed
Eg. Forensic evidence or cell from a dinosaur
What do you need for PCR?
A length of DNA mixed with 4 nucleotides. Add in taq polyemerase and the DNA will replicate many times under correct temp.
List the steps in the PCR?
- Denaturing. Heat break H bonds - DNA unzips
- Annealing. Primers added and bind to ends of single strands
- Extension step. DNA polyemerase uses free nucleotides to create complementary strands
- Repeat cycle many times to amplify large amounts of dna.
What are primers?
Short sequences that dna polyemerase recognise as start tags
You must know the nucleotide sequence just before and after the gene to be copied
Complementary forward and reverse primers are then created
What temps do the PCR steps have be?
Denaturing and breaking H bonds: 95 degrees
Annealing: 50-60 degrees
Extension: 72 degrees
What is gel electrophoresis?
Technique which separates DNA or proteins using electrical current.
Allows scientists to identify someone’s DNa
Separates pieces of DNA or proteins based on length.
How does electrophoresis work?
DNA sample placed into wells at one end of a thin slab of gel made of agarose ( or polyacrylamide) and covered in buffer. Electric current passed through the gel. Negative charged DNA attracted to anode(+)
DNA diffuses through the tiny holes of agarose. Smaller length faster and further they move. At end of run current is turned off.
How can DNA be visualised after electrophoresis?
Add ethiduim bromide- fluoresces under uv light. It’s carcinogenic.
What steps are involved in genetic fingerprinting?
- Cut DNA with restriction enzymes 2. Run DNA fragments through a gel. 3. Bands will form in the gel 4. Everyone’s DNA bands are unique.5. DNA bands are like genetic fingerprints.
What processes are involved in DNA technology?
Isolation Insertion Transformation Identification Growth/ cloning
How do you produce DNA fragments?
Retrovirus contain reverse transcriptase- turn viral RNA into DNA so it can be transcribed by host cell into proteins.
Makes DNA from RNA template
Example B cells in pancreas
How is reverse transcriptase used to make insulin?
Extract mature mRNA coding for insulin
Single strand comp cDNA is formed using reverse transcriptase on mRNA template
Single stranded cDNA used to form double strand DNA using DNA polyemerase
Why do bacteria contain restriction enzymes?
To protect themselves from invading viruses
Restriction enzymes used by bacteria to cut viral DNA
Cut at specific sites- “blunt” straight through “Sticky” staggered cut and will join another sticky end but only if cut by same rest. enzyme
Also called restriction endonuclease
List the features of a restriction endonuclease ?
Highly specific active sites
Cut about 4-8 base pairs long- called recognition sites
Palindromic- the sequence and it’s compliment are the same but reversed
Why are sticky ends important?
DNA from diff sources can be joined if they have same sticky ends- same recognition site
For this they must be cut with same endonuclease
Sticky ends joined by DNA ligase to join the sugar phos backbone
New DNA = recombinant DNA
Why are plasmids useful as vectors?
Nearly always contain antibiotic resistance genes
1 of the antibiotic resistant genes is disrupted when restrict enzyme cuts open plasmid
Other antibiotic resist gene used in selection of the correct host cells
What combinations of plasmids will occur during insertion of gene?
- Hybrid- gene inserted and permanently bonded ( IDEAL)
- Original plasmid- no change
- Gene- circle of DNA sealed together ( no plasmid involved)
All 3 are produced but only 1st is beneficial
