6 - ICH - Genomes & Gene technology Flashcards
What is another name for gene technology? Why?
Recombinant DNA technology
It involves putting the DNA from one organism into another organism
What is a transgenic organism?
Transgenic organism = Organisms that contain DNA from another organism
Summarise the method that is taken to create a transgenic organism? (6)
Isolation
- The DAN sequence making up the required gene is identified
Restriction
- DNA is cut in the correct places to remove the gene
Ligation
- Gene is inserted into a vector, usually a plasmid or virus
Transformation
- Vector carries the gene into the host, usually a bacterium
Selection
- Transformed bacteria that carry the gene are isolated
Culturing
- Bacteria carrying the gene are cultured on a large scale and the product is then harvested
State the 4 different enzymes involved in gene technology?
Restriction endonucleases
Reverse transcriptase
DNA polymerase
DNA ligase
Restriction endonuclease
- What are they
- Role
- Properties (4)
Restriction endonuclease = enzymes used to cut sections out of a DNA molecule
Found in bacteria where they break down the DNA of invading phages (type of virus) ∴ restrict viral replication
- Each restriction enzyme binds to + cuts DNA at a specific target site
- Target site is commonly 4-6 bases long and is symmetrical
- Bacterium’s own DNA is protected from attack by not having the target site, or having it hidden
- 2 strands may be cut to give blunt ends or sticky ends
- BUT sticky ends are prefered
How do bacteria protect their own DNA from being damaged by restriction endonuclease enzymes?
Their own DNA is protected from attack by not having the target site, or having it hidden
Reverse transcriptase:
- What is it
- Role
- Use
Reverse transcriptase = Enzyme used to produce single stranded DNA from mRNA
Some viruses (retroviruses e.g. HIV) contain RNA. When they infect a cell they need to convert their RNA → DNA used by the host cell - use reverse transcriptase for this
DNA polymerase:
- Role
Enzyme attach complementary DNA nucleotides to a single polynucleotide strand to produce a double stranded DNA molecule
DNA ligase:
- Role / use
Enzyme used to join okazaki fragments of DNA together
What is the use of gel electrophoresis?
To seperate DNA fragments according to their length
Describe the procedure on how to carry out electrophoresis to seperate a series of DNA fragments by size (6)
- DNA sample is cut into fragmments by restriction endonucleases
- Fragments put into wells at -ve end of a agarose gel plate using a micropipette. A dye is added to the fragments to make them easier to see
- Buffer added to surface of the gel
- Direct current is applied for a fixed time
- DNA fragments are negative due to the PO4- groups ∴ diffuse towards the +ve end of the gell tray
- Smaller fragments move faster and further than longer fragments
- Pattern of DNA bands is invisible at this time, put under blue or florescent dye which attached to DNA is added
- Compare the results to the fragment pattern of a known length of DNA - ladder
Name 3 processes that electrophoresis is used in?
Genome sequencing
Genetic fingerprinting
Genetic engineering
What is a DNA probe?
What is it used for?
DNA probe = Short single stranded piece of DNA that is labelled using a radioactive or flourescent marker and used to identify a particular DNA base sequence
- Used to identify a gene needed for genetic engineering, to aid genome sequencing and to identify allele associated with genetic disease
What does PCR stand for?
Polymerase chain reaction
What is PCR?
- What is it used for?
- Summarise it in 3 steps
Polymerase chain reaction (PCR) = Used to increase the amount of DNA for analysis in genetic fingerprinting or genetic screening
- Involves repeated replication of DNA in a test tube, doubling the amount of DNA with each replication cycle
3 Stages:
-
Denaturation
- Heat DNA to break H bonds
-
Annealing
- Lower temp and add primers which allow DNA polymerase to attach
-
Elongation
- DNA polymerase extends the primers by attaching compleentary DNA nucleotides