Section 8 - 21 Recombinant DNA technology Flashcards
What does recombinant DNA technology allow?
Genes to be manipulated, altered and transferred from organism to organism.
What is recombinant DNA?
Two different organisms that have been combined as a result of isolation, cloning and transferring.
State the stages involved in the transfer and cloning of DNA
- Isolation
- Insertion
- Transformation
- Identification
- Growth/clowing
What are three ways to perform the isolation stage of DNA technology?
- Conversion of mRNA to cDNA using reverse transcriptase
- restriction endonucleases to cut fragments containing the desired gene of DNA
- creating a gene using gene machine based on a known protein structure
How can reverse transcriptase be used in the isolation stage of DNA technology?
- select cell that readily produces the desired protein.
- these have large quantities of relevant mRNA - can be easily extracted.
- RT then used to make DNA from RNA.
- Complementary DNA (cDNA) is produced as nucleotides are complementary to mRNA.
- The enzyme DNA polymerase is used to build up te complementary nucleotides on the cDNA template.
How can restriction endonucleases be used for the isolation stage of DNA technology?
Each restriction endonuclease recognises and cuts DNA at a specific sequence of bases. - this specific site is called the recognition sequence.
- sticky ends - preferred
- smooth ends
How can the gene machine be used for the isolation stage of DNA technology?
- Determine desired amino acid sequence. - mRNA codon looked up and complementary DNA triplets worked out.
- Feed desired nucleotide bases into the computer.
- Check sequence for biosafety + biosecurity.
- Computer designs series of small, overlapping single strands of nucleotides.
- automated process, each oligonucleotide is assembled.
- Gene doesn’t have introns
- Can be replicated using polymerase chain reaction.
- sticky ends mean gene can e inserted into bacterial plasmid (vector)
What are the advantages of using the gene machine?
- any sequence of nucleotides can be produced.
- Quick (little as 10 days)
- Great accuracy.
- Free of introns.
In what two ways can you clone genes?
- in vivo - transfer into host cell using a vector
- in vitro - using polymerase chain reaction
Define - recognition site
The sequence of DNA that is cut by restriction endonucleases.
What is the enzyme DNA ligase used for?
Once the complementary bases of two sticky ends have been paired up. DNA ligase used to bind the phosphate-sugar framework of the two sections of DNA and so unite them as one.
Why are sticky ends so important?
- provide the same restriction endonuclease is used
- we can combine the DNA of one organism with that of any other organism.
How is a DNA fragment prepared for insertion?
- RNA polymerase attaches to the DNA near a gene
- Promotor - the biding site for RNA polymerase.
- The nucleotide bases of the promotor attach both RNA polymerase and transcription factors.
- This begins the process of transcription.
- At the same time the terminator region releases RNA polymerase and ends transcription.
Why is plasmid the most common type of vector used?
- Circular length of DNA
- found in bacteria
- contain genes from antibiotic resistance
- the restriction endonuclease can be used at one of these genes to break the plasmid loop.
Why is the same restriction endonuclease used during isolation and insertion?
- ensuring the sticky ends of the opened-up plasmid are complementary to the sticky ends of the DNA fragment.
Explain the process of insertion
- break plasmid at antibiotic resistance site.
- Use same restriction endonucleases.
- When the fragment is mixed with opened up plasmid they become incorporated
- join made permanently using DNA ligase
- Now known as recombinant DNA
What does transformation involve?
- plasmid and bacterial cells being mixed together in a medium containing calcium ions.
- The calcium ions and change in temp make bacterial membrane permeable.
- Plasmids can pass through into cytoplasm.
Why don’t all bacterial cells possess the DNA fragment after insertion?
- fewer than 1% of bacterial cells take up plasmids when mixed.
- some plasmids will close without incorporating fragment
- DNA fragment may join together to form own plasmid
Why are marker genes used?
To identify whether a gene has been taken up by bacterial cells.
Why three marker genes can be used in DNA technology?
- antibiotic resistant
- fluorescent proteins
- enzymes
Explain the use of antibiotic-resistant marker genes
- Replica plating - using other antibiotic-resistance gene in the plasmid - gene that is cut in order to incorporate the required gene.
- cloning cells.
- As the gene is cut it no longer produces the enzymes needed.
- problem is the cells die that contain the plasmid.
- however this identifies which contain.
how are fluorescent marker genes used?
- jellyfish - gene produces green protein. (GFP)
- gene to be cloned is transported to the centre of GFP gene
- any bacterial cell that takes up the plasmid will not be able to produce GFP
- so not fluoresce.
- don’t need replica plating as the desired cells have not been killed.
- very rapid process.
How do enzyme marker genes work?
- Gene that produces lactase
- lactase turns particular colourless substrate blue.
- transplant required gene into a gene that makes lactase.
- if plasmid with the required gene is present in the bacterial cell the colonies grown from it will not produce lactase.
- substrate stays colourless.
- remove bacteria that turns substrate blue.
What does the polymerase chain reaction do?
Method of copying fragments of DNA.
It is an automatic process making it both rapid and efficient.
what does the polymerase chain reaction require?
- DNA fragment to be copied
- DNA polymerase (taq polymerase)
- primers - short sequences of nucleotides with set of bases complementary to those at me end of each two fragments.
- nucleotides
- thermocycler
Explain the three stages of the polymerase chain reaction
- seperation of the DNA strand - place all in vessel of the thermocycler. - 95*C - two strands of DNA separate due to breaking of hydrogen bonds.
- Addition (annealing) of the primers - 55*C - primers join to comp bases at end of fragment. - provides starting sequence for DNA polymerase to being copying - also prevent strands rejoining.
- synthesis of DNA - 72*C - DNA polymerase add comp nucleotides to strands.
Repeat process so 4 strands.
What are the advantages of in vitro gene cloning?
- Extremely rapid
- can be used when only the tiniest bits of DNA are available
- doesn’t require living cells - only base sequence
What are the advantages of in vivo gene cloning?
- useful when wishes to introduce a gene to another organism - vectors - gene therapy
- no risk of contamination
- very accurate
- cuts out specific gens
- produces transformed bacteria that can be used to produce large quantities of gene products.
What is a DNA probe?
A short, single-stranded length of DNA that has some sort of label attached that makes it easily identifiable.
What are the two most popular probes used in recombinant DNA?
- Radioactively labelled probes - nucleotides with isotope 32p. - identify using X-ray film exposed by radioactivity.
- fluorescently labelled probes emit light under certain conditions.
Explain how DNA probes are used to identify particular alleles of genes
- Probe has base sequence complementary to part of base sequence of DNA that makes up the allele of the gene.
- Double-stranded DNA is being tested is treated to separate two strands.
- Strands mixed with the probe so bind to comp base sequences. - AKA DNA hybridisation
- site where probe binds is identified by radioactity or fluorescence.
When does DNA hybridization take place?
- When a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases.
- DNA must be broken to two stands.
- heat DNA until denatured
- cool to anneal bases
Explain the process of identifying whether someone possesses a mutant allele.
- Determine base sequence of mutant allele trying to locate.
- Use either DNA sequencing techniques or genetic libraries
- fragment of DNA produces with comp base sequence to desired.
- polymerase chain reaction makes copied of DNA probe.
- The probe made by attaching a marker.
- heat DNA of person so DNA separated.
- strands cooled with a mixture containing many probes
- if contains mutant then probes bine
- NDA washed clean of any unattached
- remaining hybridised DNA now fluorescently labelled with a dye attached to probe
- dye detected by shining light one fragments causing the dye to ffluoresce
What is an advantage of genetic screening?
personalised medicine
What is genetic counselling?
Advice and information are given to enable people to make personal decisions about themselves or their offspring.
Important to gage a family history.
What is a variable number tandem repeat?
DNA bases which are non-coding.
- The VNTR of every person has a unique pattern, the exception of identical twins.
- The more closely related the more similar the VNTR.
As 95% of DNA is not known to code for any characteristic.
What is gel electrophoresis used for?
To separate DNA fragments according to their size.
Explain the process of gel electrophoresis
- Place DNA fragments in agar gel
- Apply voltage across it
- The resistance of the gel means larger fragments move more slowly.
- Over a fixed period of time, the fragments spread so smallest move the furthest.
- If labelled the DNA fragment can be determined. Eg. radioactive probes.
- Place on X-ray film over agar gel.
What is a drawback of gel electrophoresis?
- Can only use DNA fragments up to 500 bases
- Larger genes and whole genomes must to cut into smaller fragments by restriction endonucleases.
Outline the 5 main stages of genetic fingerprinting
- extraction
- digestion
- separation
- hybridisation
- development
Explain the extraction stage of making a genetic fingerprint
- Seperate DNA from rest of cell
- As normally very small use polymerase chain reaction to increase the quantity.
Explain the digestion stage of making a genetic fingerprint
- Cut DNA into fragments
- Using restriction endonucleases
- These can cut close to but not within the target DNA
Explain the separation stage of making a genetic fingerprint
- gel electrophoresis separated fragments of DNA according to sie
- gel immersed in alkali in order to separate the double strands into single strands.
Explain the hybridisation stage of making a genetic fingerprint
- radioactive or Fluorescent DNA probes used to bind with VNTRs
- these probes have base sequences which are complementary to the base sequence of VNTRs - bind under specific conditions
- Process carried out by different probes
- these each bind to different target DNA sequences
Explain the development stage of making a genetic fingerprint
- X-ray film put over the nylon membrane
- the film is exposed by the radiation from probes
- the points correspond to the position of DNA fragments separated during electrophoresis
- series of bars revealed
- this pattern is unique to all individuals except identical twins.
State the uses of DNA fingerprinting
- genetic relationships and variability - paternity tests - mixed between mother and father - also variation - pop similar genetic fingerprints less genetic diversity.
- Forensic science - crime scenes
- medical diagnosis - eg. Huntington - match DNA of those with the disease and those being diagnosed.
- plant and animal breeding - prevent undesirable inbreeding during breeding programmes. - selective breeding
