Recombinant DNA technology Flashcards
What is the process of making a protein using DNA technologies
- isolation
- insertion
- transformation
4.identification
5.growth and cloning
What are the methods of producing a DNA fragment
-using reverse transcriptase
-restriction endonuclease
-gene machine
How are DNA fragments produced using restriction endonuclease
-DNA is cleaved at a restriction site
-restriction endonuclease cuts out the DNA fragments
-staggered cuts leave sticky ends which are several nucleotide bases long
-Any piece of DNA cut with the same restriction site will have complimentary sticky ends
-Complimentary bases of the sticky ends pair up
-DNA ligase joins sugar phosphate backbone
-Allows us to join DNA from one organism to another
How are DNA fragments produced using reverse transcriptase
-Strand of mRNA from the beta cells of human pancreas coding for the production of insulin
-mRNA acts as a template for the production of a single stranded complimentary copy of cDNA using reverse transcriptase
-cDNA is isolated by hydrolysis of the mRNA with DNA helicase
-Double stranded DNA is formed on the template of the cDNA using DNA polymerase resulting in a copy of the human insulin gene
How are DNA fragments produced using the gene machine
-Desired protein with amino acid sequence, mRNA sequence and DNA sequence
-nucleotide base sequence into computer
-biosafety, biosecurity and ethics checked
-computer designs oligonucleotides
-oligonucleotides assembled one nucleotide at a time
-oligonucleotides joined to form gene
-PCR replicates gene
-gene into plasmid using sticky ends
-gene check using sequencing techniques
What does in vivo mean
transferring the fragments to a host cell using a vector
What does in vitro mean
using the polymerase chain reaction
Describe the process of in vivo gene cloning
-DNA fragment is produced from restriction endonuclease
-RNA polymerase and transcription factors binds to the promoter region of the DNA fragment
-Terminator is added to stop transcription at appropriate time
-Same restriction endonuclease used to cut fragment is used to cut plasmid at antibiotic resistant gene which produces complimentary sticky end
-plasmid is transformed into the bacteria using calcium irons and changing the temperature
-identify uptake of gene/plasmid using R plasmid which carry the gene for resistance to two antibiotics, ampicillin and tetracycline
-Determine if the gene is present using a marker gene
Describe the process of PCR
-DNA fragments, primers and DNA polymerase placed in thermocycler and heated to 95 to break hydrogen bonds separating the strands
-cool to 55 so primers can anneal to DNA at complimentary bases
-heat to 72 so free nucleotides can bind to complimentary base pairs and DNA polymerase can form phosphodiester bonds
What are the advantages of in vitro cloning
-it’s extremely rapid
-it does not require living cells
What are the advantages of in vivo cloning
-it is particularly useful where we wish to introduce a gene into another organism
-it involves almost no risk of contamination
-it’s very accurate
-it cuts out specific genes
-it produces transformed bacteria that can be used to produce large quantities of gene products
What does PCR require
-DNA fragment
-DNA polymerase
-primers
-nucleotides
-thermocycler
What is a primer
Short sequence of nucleotides that have a set of bases complimentary to those at one end of each of the two DNA fragments
What is a thermocycler
A computer controlled machine that varies temperatures precisely over a period of time
DNA probe
short single stranded length of DNA that has a label attached to it that makes it easily identifiable
How are DNA probes used
-Isolate DNA from bodily fluid sample
-denature the single strand of DNA
-combine with DNA probes that are complimentary
-DNA probes will bind to the gene of interest if it’s present in the sample
What are the two most common types of DNA probe
-Radioactively labelled probes
- fluorescently labelled probes
How are radioactive DNA probes identified
Using X-ray film that is exposed by radioactivity
How are fluorescent DNA probes identified
Emit light under certain conditions, for example when the probe has bound to the target DNA sequence
DNA hybridisation
To measure the degree of difference between two strands of DNA. Can be used to compare someone’s DNA to a certain gene to see if they have it
How are specific alleles located using DNA probes and DNA hybridisation (mutant allele that causes disease)
-Determine the sequence of nucleotide bases of the mutant allele
- Make a DNA probe that has bases complimentary to the mutated gene
-Separate the double stranded DNA from the sample to make it single stranded using DNA helicase or heat
-Mix separate strands of DNA sample with DNA probe and cool
-DNA probe will bind to the complimentary base sequence of the mutated gene on the DNA sample (DNA hybridisation)
-wash away unattached DNA probes
-site where the probe has bound is identified by either the radioactivity or fluorescence that the probe emits
genetic counselling
allows people to make informed decisions about themselves or their offspring by providing advice and information
genetic screening
can determine the probability of a couple having offspring with a genetic disorder
genetic fingerprinting
process that relies on the fact that the DNA of individuals is unique and contains repetitive non-coding bases called VNTRs
What are VNTRs
They are non-coding and inherited from parents. They are unique in every indivual expect twins
Gel electrophoresis
used to separate DNA fragments according to size
How does gel electrophoresis work
-DNA fragment is placed on agr gel and voltage is applied along it
-resistance of the gel means that the larger the fragments, the more slowly they move
-over a period of time the smaller fragments mover further
-If the fragment is labelled with a probe it can be determined with a x-ray sheet
What are the 5 stages of making a genetic fingerprint
-extraction
-digestion
-separation
-hybridisation
-development
How are the 5 stages of genetic fingerprinting carried out
-DNA is extracted from the sample
-Restriction endonucleases cut the DNA into fragments
-They are separated using gel electrophoresis and transferred to from the gel to nylon membrane
-DNA probes are added to label the fragments and they attach to specific fragments
-membrane with radioactively labelled DNA fragments is placed onto an X-ray film
What are the uses of DNA fingerprinting
-crime scene investigation
-resolve questions of paternity
-medical diagnosis
How is DNA accepted and functioning by different species
the genetic code is universal
palindromic
when you read the unpaired bases both from left to right the two sequences are opposites of one another
What are advantages of using the gene machine
-any sequence of nucleotides can be produces in a short time
-accurate
-the artificial genes are free of introns and other ‘non’ coding DNA
What is the importance of sticky ends
Complimentary sticky ends from different organisms can be joined using DNA ligase
How do R-plasmids work in in vivo gene cloning
-all the bacteria cells are grown on a medium containing ampicillin
-bacterial cells that have taken up the plasmid will have acquired the gene for resistance
-these bacterial cells are able to break down the ampicillin and survive
-the cells that have not taken up he plasmids will not be resistant and die
Marker genes
Involve using a second, separate gene on the plasmid which is easily identifiable
Why are marker genes easily identifiable
-May be resistant to an antibiotic
-May produce a fluorescent protein that is easily seen
-Produce an enzyme whose actions can be identified
Antibiotic resistance marker genes
replica plating
-requires tetracycline which was cut
-no longer produce the enzyme that breaks it down
-no longer be resistant
-can therefore identify these bacteria by growing them on a culture that contains tetracycline
Fluorescent markers
-transfer of a gene from a jellyfish
-produces a green fluorescent protein (GFP)
-Any bacterial cell that has taken up the plasmid with gene that is to be cloned will not be able to produce GFP
-Results can b obtained by looking under a microscope
vector
transfers genes from one organism into another
Enzyme markers
-gene that produces the enzyme lactase
-lactase will turn a particular colourless substrate blue
-required gene is transplanted into the gene that makes lactase
-If the plasmid with the required gene is present it won’t produce lactase- won’t change colour
polymerase chain reaction
method of copying fragments of DNA
