Gentetic engineering Flashcards
Define genetic engineering/ DNA technology
Aims to remove a desired gene and transfer it to another organism where it can be expressed. This means that the required protein can be synthesised within the new organism.
Why do we use DNA technology/ genetic engineering?
Sometimes humans cant make certain proteins because of faulty genes like insulin so this technique allows for the mass production of a protein to treat medical conditions. It allows us to study gene function Allows us to treat genetic conditions/ diseases via gene therapy
How is it possible that a gene from ONE organism can be transferred into another completely separate species and expressed in that organism (GMO
Genetic code in all organisms is universal The process of making proteins from the genetic code is universal- mechanisms of transcription and translation is the same in ALL organisms
What are the 2 main ways of genetic cloning
- In vivo - cloning is done using a living organism – 5 stages (to be discussed) OR 2. In vitro – cloning does not involve a living organism – in another lesson
What are the outcomes of gene cloning
-rDNA (recombinant DNA) -if it involves a living organism: GMO (genetically modified organism) This expresses the desired gene and will produce the protein
What are the 6 stages of gene cloning
- Isolation of the DNA that contains the gene – 3 ways 2. Amplification – this step not taught at AQA 3. Insertion – inserting gene into plasmid (vector) 4. Transformation – inserting vector into a suitable host (bacteria) 5. Identification – seeing which bacteria express the gene of interest 6. Growth/cloning – mass production as the bacterial (host cells) reproduce
STEP 1: ISOLATING THE GENE what are the 3 ways you can do this
- Using reverse transcriptase 2. Using restriction endonucleases (enzymes) 3. Creating the gene in a gene machine – based on KNOWN protein structures
what are retroviruses?
A group of viruses e.g. HIV The coded genetic info in retroviruses is in the form of RNA
Isolating target gene: method 1: Reverse Transcriptase DRAW A DIARGRAM/ DESCRIBE THIS PROCESS
Use a cell that express large amounts of your desired protein e.g. insulin (β cells of the pancreas) mRNA will be present which codes for insulin e.g. UGCUUA Use an enzyme called reverse transcriptase (from a retrovirus) to converts mRNA to cDNA (complementary DNA). mRNA acts as a template to build cDNA - ACGAAT Make a double stranded molecule by using DNA polymerase
ISOLATING TARGET GENE STEP 1, METHOD 2: How do bacteria use restriction endonucleases
Bacteria are frequently infected with viruses – that inject their DNA into them. Bacteria then use restriction endonucleases to chop of the viral DNA and makes it non-functional.
What makes each restriction endonuclease unique?
They all recognise a different recognition sequence so need different shaped active sites
Method 2: Restriction enzymes/endonucleases Describe this process
Used to cut the gene out directly They each cut DNA at specific sites which is 4-6 bases long– called a recognition sequence They can cut straight or staggered ends Straight – cuts occur between 2 opposite base pairs, leaving blunt edges Staggered – cuts occur that are not opposite base pairs – sticky end
What are restriction sites
The sites recognised by the endonucleases are called restriction sites and these are usually in the form of palindromes (reads the same both ways, like “Hannah” or “Madam“ - symmetrical)
STEP 1, METHOD 2: Restriction endonucleases whats the difference between blunt and sticky ends
Enzymes may cut at the SAME place in BOTH strands creating blunt ends e.g HpaI or at DIFFERENT places in the 2 strands leaving so-called sticky ends (staggered unpaired bases) as in EcoR1 or HindIII. Sticky ends important for cloning
STEP 1, METHOD 2: Restriction endonucleases What are sticky ends
Have exposed bases that will form hydrogen bonds with complementary sticky ends from other DNA molecules cut with the SAME enzyme
STEP 1, METHOD 2: Restriction endonucleases What are blunt ends
Some restriction enzymes, such as Hpal, cut the DNA strands at positions directly opposite one another, giving blunt ends to the fragments
Method 3: The Gene Machine Describe the process
- Determine the desired protein’s amino acid seq therefore mRNA seq, to determine complimentary DNA triplets.
- Feed desired DNA seq to computer to check for biosafety,biosecurity and so it meets teh standards and ethical requirements
- Computer designs oligonucleotides to be assembled into a gene
- Gene replicated with PCR to make sticky ends
- Check for sticky ends
what are advantages to gene machine?
Any gene can be made in a short amount of time- about 10 days Very accurate Artificial genes are free of introns and other non-coding DNA and so can be transcribed and translated by prokaryotic cells
IN VIVO Transformation: Describe the process
Complementary sticky ends
created by cutting the DNA
from each species with the
same restriction enzyme
Complementary bases on the sticky ends of the DNA from the different species are attracted to one another
Hydrogen bonds
form between the
bases and the
enzyme DNA ligase
seals the sugar-phosphate backbone
of the DNA molecule
whats the next main step in recombinant DNA technology, after forming DNA fragments and what ways can you do this?
You need to amplify the DNA fragments so that there are more.
- In vitro: PCR (polymerase chain reaction)
- In Vivo
Describe in vivo- transformation
1) DNA cut using restriction endonucleases to create fragments with sticky ends
2) A promoter and terminator region are added to allow transcription
3) The same restriction endonuclease is used to cut open the plasmid (DNA loop in bacteria, which adds as a vector) to allow complem., sticky ends
4) The enzyme DNA ligase used to incorporate the DNA fragments into the plasmid-recombinant DNA is formed
5) Recomb. plasmid transferred into bacteria via heat shock or calcium ions to increase membrane permeability
what are the problems with in vivo- transformation and how do solve these
- not all plasmids take up the foreign DNA fragments
- not all recombinant plasmids will be taken up by the bacterial cells
-Can identify which bacterial cells have taken up the recombinant plasmids through marker genes
how are marker genes involved in e.g. identifying if a plasmid is taken up
ANTIBIOTIC RESISTANCE MARKER GENES
- plasmids contain antibiotic resistance genes
- bacteria are grown on agar plates containing antibiotics
- if bacteria have taken up the plasmid they’ll survive as they will contain anibiotic resistance genes
- bacteria will die if they haven’t taken up the plasmid as they don’t contain antibiotic resistant genes
how can marker genes be used in e.g. identifying if the plasmids are recombinant
FLUORESCENCE
- DNA fragments inserted in the middle of a marker gene e.g. GFP gene which encodes fluorescence
- GFP will no longer be made so the plasmids that don’t fluoresce are recombinant and those that do are non. so haven’t taken up the foreign DNA frag.
what is gene therapy and describe the process
mechnanism by which genetic disorders can be cured or treated by masking the effect of the faulty allele with the insertion of the functional allele.
1) Healthy allele isolated and inserted into cells using vectors
2) If mutant allele is recessive , a dominant allele is inserted and if the mutated allele is dom, DNA is inserted into middle to silence it
what are the 2 main types of gene therapy
somatic and germline
somatic= alleles in body cells are altered and isn’t passed on to offspring
germline= alleles in sex cells are altered and are passed on to offspring but is considered unethical due to concern over designer babies or safety of gene therapy
what is genetic fingerprinting
- method used to produce a specific pattern of DNA bands from an individual’s genome
- they use VNTRs
what are VNTRs
- variable number tandems repeats
- short, repeating sequences of DNA in non-coding regions
- in every individual, they vary in length and in the number of repeats so the probability of 2 individuals having the same VNTRs is v low
Describe the process of DNA fingerprinting
) extraction of the DNA of interest and amplification of PCR
2) digestion using the specific restriction endonucleases into DNA fragments
3) separation of DNA fragments by gel electrophoresis
- DNA fragments move towards the negative electrode
- smaller fragments move further down the gel
- different sized fragments are separated into bands
4) VNTRs are hybridised at specific complemen. base seq. with DNA probes
5) Gel develops- pattern of bands can be visualised by putting gel on xray film as probes emit radiation which reveals the positions
how can we use bands from gel electrophoresis for DNA profiling
- can determine genetic relationships by looking at how similar the banding patterns are
- can use in forensics for comparing DNA profile of suspect with those found at the crime scene
- can use in medical diagnosis- identify individuals at risk of developing particular diseases e.g Huntingtons
- Can use in plant/animal breeding- used to prevent inbreeding by not breeding those with similar patterns
what are DNA probes and why are they used
- DNA probes are short sections of DNA that are complementary to a known DNA seq. e.g. mutated allele
- labelled with a fluorescent or radioactive tag
- mainly used for genetic screening: study of an individual’s DNA to identify whether they possess a mutated allele that causes a particular disease
how do DNA probes work
- the labelled DNA probe is mixed with denatured DNA samples from an individual
- if the individual has the mutant allele, the probe will bind to the complementary base seq. in one DNA strand- hybridisation
- the hybridised DNA is detected using the radiation and fluorescence
