Videos- Recombinant DNA Tech Flashcards
3 ways to isolate target genes
1) Restriction enzymes
2) Reverse transcriptase
3) Artificial synthesis of gene
What does palindromic mean?
Sequence reads same forward and backwards
What does reverse transcriptase do?
Enzyme that does transcription backwards- mRNA➡️ cDNA
What’s cDNA?
Complementary DNA that has no introns made using reverse transcriptase
What’s a digonucleotide?
Nucleotides joined together during the process is artificial synthesis of a gene (max 25)
What’s the process of making a gene from scratch? (Artificial synthesis gene)
1) Use gene machine.
2) Form digonucleotide, join multiple digonucleotides together to make a synthetic gene.
How to isolate a target gene using restriction enzymes?
Restriction enzymes cut DNA at restriction sites. Leaves DNA with sticky ends.
What’s a restriction site?
Specific palindromic site
3 main steps in forming a transgenic bacteria
1) Isolate target gene
2) Inset gene into vector
3) Insert vector into bacteria
4 things needed for isolating target genes
1) Promoter region
2) Terminator gene
3) Sticky ends
4) Marker genes
What’s a vector?
Something used to move DNA from one place to another
2 types of vectors
Virus- bacteriophage
Plasmid- double stranded loop of DNA, transfers genes between bacteria
How do you insert a gene into a vector?
Use restriction enzyme to cut plasmid =complementary sticky ends
DNA ligase reforms phosphodiester bonds
Forms
How do you insert a gene into a vector?
Use restriction enzyme to cut plasmid =complementary sticky ends
DNA ligase reforms phosphodiester bonds
Forms recombinant DNA
How do you insert the vector into the bacterium?
Ice cold CaCl2 and heat shock it
What’s formed when a vector is inserted into a bacterium?
Transgenic organism
Why are marker genes used in recombinant DNA?
To identify the transformed bacteria
What’s a marker gene? (According to tailored tutors)
Easy to identify
Paired with target genes to see whether the vector has been incited properly
2 examples of marker genes
1) UV fluorescence
2) Antibiotic resistance
What does the polymerase chain reaction do?
In vitro amplifies DNA, makes lots of copies
What do you need for PCR? (polymerase chain reaction)
DNA sample
Free DNA nucleotides
Primer
DNA polymerase
What’s the method of PCR?
1) Heat to around 95 degreesC
2) Cool to around 50 degreesC
3) Heat to around 70 degreesC
4) Repeat.
What’s the reason for the first step in PCR? (Heating to around 95 degreesC)
Break hydrogen bonds
Make DNA single stranded
What’s the reason for the second step in PCR? (Cool to around 50 degreesC)
Allows primer to bind
Complementary base pairing
DNA double stranded
DNA polymerase can bind
What’s the reason for step 3 in PCR? (Heat to around 70 degreesC)
DNA polymerase adds complementary nucleotides
Makes phosphodiester bonds
What’s the reason for repeating the process of PCR? (4th step)
To make as much DNA as possible, each cycle doubles the amount of DNA
What is electrophoresis?
Using electricity to seperate DNA fragments by length
What’s the process for electrophoresis?
Attach fluorescent label/stain DNA fragment
Put marked DNA fragments in well at anion
Turn on current
DNA is negative so is attracted to cation
Smaller fragments move faster so further as there’s less resistance.
How do you calibrate your scale for electrophoresis?
Using known lengths of DNA
Summary of gene technology process
1) isolate target gene
2) insert gene into vector
3) insert vector into bacterium
4) identify transgenic bacteria
5) culture transgenic bacteria
6) extract and purify protein
Who do you culture your transgenic bacteria?
Transcribe and translate recombinant DNA
Make proteins of target gene
What’s the purpose of recombinant DNA and gene technology?
To make a protein from your target gene
E.g. Insulin
What is gene therapy?
Changing faulty alleles that cause genetic disease
How do you change a faulty allele if the allele at fault is dominant? (Process)
Silence dominant allele
1) use vector to add DNA fragment into dominant allele
2) dominant allele isn’t transcribed
3) by default recessive allele is expressed, (transcribed and translate)
How do you change a faulty allele if the allele at fault is recessive? (Process)
Sufferer is homozygous
1) use vector to add functional allele to DNA- has to be added in right locus
2) dominant allele expressed
What is germ line gene therapy?
Changing alleles of gametes
What so somatic gene therapy?
Changing alleles of body cells
How do somatic and germ line gene therapy compare with how they affect future offspring?
Germ line- offspring inherit changes
Somatic- offspring don’t inherit changes
What are 4 possible problems of gene therapy?
1) alleles inserted into wrong locus
2) silence wrong gene e.g. Tumour suppressor gene which would cause cancer
3) gene over expressed
4) used for non-medicinal reasons e.g. Designer babies or makeup
What are the uses of genetic modification in agri- e.g. Soya beans?
Express protein from bacteria
Protein toxic to insects
Fewer insects on plants
What are the advantages of genetically modifying soya beans?
Use less chemical pesticides
More effect food chain
How and why is golden rice genetically modified?
Gene from corn p,ant inserted into rice plant
Allows VitA expression
What’s the advantage of genetically modifying golden rice?
Prevents blindness from VitA deficiency
What are 3 disadvantages of genetically modifying plants in agriculture?
1) monoculture of crop with low genetic diversity- susceptible to disease/enviroment
2) buy new seed every year- termination gene
3) decreased biodiversity
What does industry and research use genetic modification for?
1) making enzymes
E.g. Re in (cheese) and lipase
2) transform pathogens to treat disease- attack other pathogens without infecting humans
What’s the advantage of genetically modified enzymes?
Reduced energy and cost
Fast and cheap production
What’s the advantage of genetically modified pathogens?
Treats disease
Pathogens won’t develop resistance
Reduces suffering
What are the disadvantages of genetically modified pathogens?
Could mutate and infect humans
Could be used in war as bio weapons
What does medicine ‘pharming’ use genetic modification for?
Transform bacteria to express proteins
Transform mammals to produce useful products in their milk
What’s the advantage of genetic modification in medicine?
Make human proteins
Cheaper and easier than making synthetic proteins
What are the disadvantages of genetically modified medicine?
Possible unexpected problems e.g. Cancer in mammals
Using animals as a commodity
