- polymerase chain reacton- replicating many copies of DNA in a test tube- carrued out in a thermocyclyer
- like semi conservative replication in cells- but heat is used to break open hydrogen bonds in double helix- primers are used to maek the end and beginning section of DNA which is being copied
- primers are short single stranded nucleic sequences - DNA polymerase used as well.- special heat stable DNA polymerase because heat is used to break strands.
- Process: DNA is copied and placed in a test tube with DNA p,primers and DNA nucleotides then...
1) mixture heated to 93-hydrogen bonds break-two strands of DNA -single stranded
2) DNA cooled 55-primers can attach to the end of DNA sequences that are being copied.
3) DNA heated 72-DNA p (optimum temp of enzyme) joins new nucleotides on DNA strands= 2 identical strands
Uses of PCR
- making copies of DNA from a single embryo cell so enough DNA can be extracted for genetiic analysis
- making copies of small amounts of DNA which can be extracted from fossils and mumified remains
- making many copies of DNA found in crime scene investigations - analysed by DNA finger printing
Making recombinant DNA
Genetic engineering is a collection of techniques by ehich genes are altered or transfered from one organism to another- called recombinant DNA. genetic engineering involves obtaing and isolating the gene to be transferd, transfering the gene into new cells and identifying the modified cells.
Isolation of a gene
- insulin production came from pigs to treat diabetes (one amino acid difference)- nowadays human insulin cann be inserted into a bacterial cell - the bacteria grow in large numbers and hinsulin is extracted -no immune respone.
- first human insulin gene needs to be otained by: cutting the gene out of the human DNA using a restriction enzyme (cut DNA when they find a specific base sequence) - leaves sticky ends, made form mRNA using reverse transcriptase to make a DNA copy from its RNA - make single stranded DNA copy of mRNA (cDNA)-made into double stranded, gene machine because we can work out the DNA base sequence for human insulin.
inserting DNA into new cells
- a vector is used to transfer the isolated gene into a new host cell ( common vector-plasmid)- the DNA in the plasmid is cut open using a restriction enzyme sometimes its the sme one used to isolate the gene if not nucleotides are added to the cut plasmid and the isolated gene so they have matching sticky ends
- This gene is inserted into the cut plasmid using DNA ligase
- Bacterial cells and plasmids mixed together bacteria are treated so they take up modified plasmid-recombinant DNA
using genetic markers
- plasmid contains antibiotic resistance gene - resistance to a certain antibiotic.
- scientists mix bacterial cells with antibiotic (that marker gene gives resistnace)- killing bacteria that doesnt contain plasmid- marker genes can also code for other charcteristics (production of enzyme)
Gene Probe and Gene Libaries
- a gene probe is a single stranded sequence of DNA or RNA which is complementary to the nucleotide sequence of a specific gene.- specifically labelled i.e radioactive/fluroscent - so it will bind to complementary base sequence - which can be identified.
- Using a Gene probe:
- DNA to be tested is extracted- if a bigger sample needed PCR used.
- DNA is digested into pieces using a restriction endonuclease
- DNA fragments seperated using gel eletrphoresis the DNA is placed in a well in a gel- electric current applied. DNA fragments move towards the + electrode - smaller pieces move quicker through the gel - DNA pieces seperated according to size.
- gel containing the DNA is treated with an alkali so the DNA becomes single stranded= single stranded DNA is then transfered to a membrane
- gene probe added to the membrane and time is allowed for it to bind to its complementary sequence
- membrane washed- if probe is radioactive - membrane is placed against a piece of photographic film - if probe has bound to the DNA on the membrane 'fogging' will occur alternatively if the probe is florescent - it will show up under UV.
- a set of thousands of DNA fragments from one organism
- the DNA of the the organism is cut using a restriction endonuclease- each fragment is inserted into a plasmid to make recombinant plasmid.which are then inserted into bacterial cells
- alternatively mRNA is isolated from cells - reverse transcriptase is used to make a DNA copy of mRNA - each piece of DNA is added to a seperate plasmid and inserted into a bacterial cell
- each clone of bacteria is stored in a seperate well in a multi well dish- they may be stored , frozen for a ong time- later is gene required DNA of bacteria can be tested using a gene probe
- locating a gene can be done by testing a a clone of bacteria using an antibody specific to the protein produced by a gene of interest
- gene libaries are being used to store genetic material- if the gene libary contains all the DNA from the organism - representive libary - meaning that a specific gene from the organism can be found in at least one of the fragments.