Gene technology Flashcards
Stages in producing a protein using DNA technology
Isolation of DNA fragments that produce protein
Insertion of DNA into vector
Transfer of DNA into host cell
Identification of cells that successfully took up DNA using host markers
Growth and cloning
How to use reverse transcriptase to convert mRNA to cDNA
Cell that produces protein is selected as they have large quantity of the mRNA. Reverse transcriptase converts mRNA to cDNA.
DNA polymerase builds up complementary nucleotides on cDNA to produce double stranded DNA
How to use restriction endonucleases to produce DNA fragments
Cuts gene out leaving sticky ends
How to use gene machine to produce DNA fragments
Amino acid sequence is determined and then mRNA codons are looked up and comp DNA triplets worked out.
Sequence of nucleotides fed into computer and assembled by oligonucleotides. Polymerase chain reaction constructs complementary strand and copies gene many times.
Inserted into plasmid using sticky ends
Advantages of gene machine
Any sequence can be produced in short time.
Free of introns so can be transcribed by prokaryotes
Preparation of DNA fragment and plasmid for insertion into vector
Promotor and terminator gene sequence added for attachment of RNA polymerase and transcription factors and for stopping of transcription.
Same restriction enzyme that cut out gene cuts open plasmid to create complementary sticky ends and joined using DNA ligase.
Why do some cells not take up plasmid
Only 1% take up plasmid with desired gene.
Some plasmids close together again.
DNA fragments forms its own plasmid.
How to find which bacterial cells taken up plasmids (but not necessarily gene)
Bacterial cells grown on medium containing ampicillin and therefore bacterial cells that taken up plasmid will have gene for resistance. These can break down ampicillin and survive and others will die.
How to identify cells that taken up plasmid but no gene and eliminate them
Use of a marker gene (fluorescent, antibiotic resistance, enzyme action).
How to use antibiotic resistance marker gene to identify if gene has been taken up
Incorporate gene into plasmid in the middle of antibiotic resistance gene so it stops producing enzyme. Use replica plate to identify to not kill original.
How to use fluorescent marker gene to identify if gene has been taken up
Bacterial cell that taken up plasmid will not be able to produce GFP. Retain those that do not fluoresce
How to use enzyme marker gene to identify if gene has been taken up
Gene that produces lactase that turns colourless substrate blue. Gene transplanted into lactase gene. Bacterial cells that taken up gene will not change colour of colourless substrate to blue when grown on it.
How are plasmids inserted into bacteria
Calcium ions and heat shock make membrane permeable
Primer
Short sequence of nucleotides that have set of bases complementary to those at one end of each of two DNA fragments
Separation of DNA strand in PCR
DNA fragments, primers and DNA polymerase played in thermocycler and at 95C so hydrogen bonds break
Annealing of primers in PCR
Mixture cooled to 55C so primers anneal to complementary bases at end of DNA. Provide starting point for DNA polymerase to begin copying and prevent strands re-joining.
Synthesis of DNA in PCR
Temp increased to 72C and DNA polymerase adds complementary nucleotides along each separated DNA strand until reaches end of chain.
How does PCR loop
Thermocycler increases temp to 95 at the end to break hydrogen bonds of copies to repeat again.
Advantages of PCR
Extremely rapid (but will also clone other random DNA) Does not require living cells so no culturing.
Advantages of in vivo gene cloning
Vectors used so can easily introduce gene into organism.
No risk of contamination as only complementary sticky ends cut by same restriction enzymes will be taken up by plasmid
DNA probe
Short, single stranded length of DNA that has a label that makes it identifiable with base sequences that are complementary to base sequence of target DNA.
How DNA probe works overview
DNA probe has complementary base sequences to target DNA.
Extract DNA and add restriction enzymes.
Separate DNA into fragments using electrophoresis.
Separate two DNA strands to expose bases.
Complementary DNA probe hybridises with gene.
Check for marker.
How DNA hybridisation works in DNA probing
Heating DNA until double strand separates and then when cooled, complementary sections (probe and DNA) anneal.
Locating specific alleles of genes
Extract DNA and add restriction enzymes.
Separate DNA into fragments using electrophoresis.
Treat DNA (heating) to form single strands and expose bases.
Complementary DNA probe hybridises with gene.
Check for fluorescence or use X-ray film to find bound probe.
Advantages of genetic screening using probing
Many different DNA probes can be fixed and so can test DNA simultaneously for many different disorders.
Mutated tumour suppressor genes can be identified so people can make informed decision also.
Personalised medicine.
Genetic fingerprinting
Non-coding bases of DNA has VNTRs (variable number tandem repeats) which are different for each individual in number and length.
Gel electrophoresis
Separates DNA fragments by size. Larger fragments, slower they move and so smaller fragments move further.
Gel electrophoresis - digestion
DNA cut into fragments by restriction enzymes
Gel electrophoresis - separation
DNA are separated by size according to gel electrophoresis under voltage and then gel immersed in alkali to separate strands.
Gel electrophoresis - hybridisation
DNA probes which complementary base pair sequence to VNTRs bind with VNTRs.
Gel electrophoresis - development
X-ray film exposed by radiation from proves and positions are located visually
Series of bars revealed
