✅21 - Recombinant DNA Techniques Flashcards
What is recombinant DNA?
When DNA from two different organisms is combined and grown in microorganisms to form specific proteins
What are transgenic organisms?
Ones the have been genetically modified
What stages does making a protein using gene transfer and cloning involve?
Isolation Insertion Transformation Identification Growth/cloning
What is isolation?
The DNA fragments are isolated that have the gene for the desired protein
What is insertion?
The DNA fragment is inserted into a vector
What is transformation?
The transfer of DNA into suitable host cells
What is identification?
The host cells are identified that have successfully taken up the gene using gene markers
What is growth/cloning?
The population of host cells is cloned
What are the methods of producing DNA fragments?
Conversion of mRNA to cDNA using reverse transcriptase
Using restriction endonucleases to cut fragments containing the desired gene from DNA
Creating the gene in a gene machine, usually based on a known protein source.
How can reverse transcriptase be used to produce DNA fragments?
A cell that readily produces a protein is selected
These cells have large quantities of relevant mRNA which is more easily extracted
Reverse transcriptase is then used to make DNA from the RNA, known as cDNA (Complementary).
To make the other strand of DNA, DNA polymerase is used to build up the complementary strand
What are restriction endonucleases?
Enzymes that cut up viral DNA
What are blunt ends?
When restriction endonucleases cut DNA between two opposite base pairs, leaving two straight edges
What are sticky ends?
When restriction endonucleases cut DNA in a staggered way, leaving an uneven cut
What is a palindrome sequence?
When the sequences are opposite to each other in sticky ends
How are genes prepared for a ‘gene machine’?
The desired sequence of nucleotide bases of a gene is determined from the desired protein and the amino acid sequence determined. From this, the mRNA codons are looked up and complementary DNA triplets worked out. The desired sequence of nucleotides are then fed into a computer.
What are sequences check for before being fed into the computer of the gene machine?
Biosafety and biosecurity to ensure it meets international standards as well as ethical requirements
What are oligonucleotides?
A series of small, overlapping single strands of nucleotides which can be assembled into the desired gene.
What does the gene machine do with the oligonucleotides produced?
They are joined together to make a gene which doesn’t have introns or non-coding DNA. The gene is then replicated using PCR
How are genes from the gene machine inserted into bacterial plasmids?
Using sticky ends
What does the bacterial plasmid act as in the gene machine?
The vector
What is in vivo cloning?
Transferring fragments to a host cell using a vector
What is in vitro cloning?
Using PCR
What is the role of DNA ligase?
To bind the phosphate-sugar backbones of two sections of DNA at sticky ends and bind them
What is a promoter?
The binding site for RNA polymerase and transcription factors for transcription of a gene to occur
What is a terminator?
A region of DNA that releases RNA polymerase and ends transcription
What is a vector used for?
Transporting the DNA into the host cell
What are plasmids?
Circular lengths of DNA found in bacteria which are separate form the main DNA
How are the plasmids reintroduced into bacterial cells?
The plasmids and bacterial cells are mixed with Ca2+ ions and the temperature is changes, making the bacterial membrane permeable.
Why don’t all the bacteria end up with the desired proteins produced when vectors are added?
Only a few bacteria take up the plasmids when they are mixed
Some plasmids will have closed up again without incorporating DNA fragment
Sometime the DNA fragment ends join together to form its own plasmid
How can the bacteria containing the desired genes be identified?
All the cells are grown in a medium that contains the antibiotic ampicillin
Cells that have taken up the plasmids will have acquired the gene for ampicillin resistance
These cells are able to break down the ampicillin and therefore survive
The bacterial cells that have not taken up the plasmids die.
What can marker genes be used for?
Identifying whether a gene has been taken up by bacterial cells
What can make marker genes identifiable?
Antibiotic resistance
Fluorescent protein
May produce an enzyme whose action can be identified
What are the stages of PCR?
Separation of the DNA strand, denaturation
Annealing of primers
Synthesis of DNA
What happens in denaturation?
Placed in thermocycler at 95 degrees causing hydrogen bonds to break
What happens in annealing of primers?
Mixture is cooled to 55 degrees causing the primers to join to the complementary DNA. They provide the starting sequences for DNA polymerase to begin copying.
What happens in synthesis of DNA?
The temperature increased to 72 degrees, DNA polymerase adds complementary nucleotides along each DNA strand
What are primers?
Short sequences of nucleotides that have a set of bases complementary to those at one end of each of the DNA fragments
What are the advantages of in vitro cloning (PCR)?
It is very fast - within hours billions of copies can be made of a gene.
Can be done with minute amounts of DNA
Does not require living cells
What are the advantages of in vivo cloning (using vectors)?
Particularly useful when we want to introduce a gene into another organism
No risk of contamination of different genes
Very accurate
Cuts out specific genes
Produces transformed bacteria which can be used to produce large quantities of gene products
What are the risks of recombinant DNA technology?
Impossible to predict ecological consequences of genetically engineered organisms in environment
Genes may pass on to other organisms, eg through viruses
DNA manipulation has consequences for metabolic pathways
All genes mutate
What are the benefits of recombinant DNA technology?
Microorganisms can be modified to produce a range of useful substances
They can be used to control pollution eg digesting oil slicks or gases
GM crops have financial and environmental advantage, making plants more tolerant to extremes and better yield
What are DNA probes?
Short, single stranded lengths of DNA that have some sort of label attached
What are examples of DNA probes?
Radioactively labelled and fluorescent labelled
What are radioactively labelled probes?
Made up of nucleotides with the isotope 32P. Identified using an X ray film that is exposed by radioactivity
What are fluorescently labelled probes
They emit light under certain conditions, such as when the probe has bound to the target DNA
How are probes used to identify particular alleles of genes?
A DNA probe is made that has base sequences that are complementary to target DNA
DNA being tested is separated into two strands
Strands mixed with probe, binds to complementary sequence
The site where the probe binds is identified
What is DNA hybridisation?
When a section of DNA or RNA binds with the complementary base sequence on a single stranded section of DNA
What is the process for identifying alleles or mutations?
Produce DNA probe PCR to amplify DNA Heat DNA to denature it Cool and add probes (anneal) Wash DNA to remove unattached probes Detect dye or fluoresence
What can genetic screening be used for?
Oncogenes and mutations leading to genetic disorders
What is personalised medicine?
Advice and healthcare based on an individual’s genotype
What are examples of personalised medicine?
Painkillers - half population have non functioning enzyme essential for some painkillers
Vitamin E - can reduce risk of cardiovascular disease for some genotypes but increase it for other
What is genetic counselling?
A form of social work where advice is given to help people make decisions on either themselves or their offspring
What can geentic screening be used to detect in cancer patients?
Oncogene mutations to determine most effective treatment
Gene changes which indicate which treatment will benefit the patient best
A single cell which may lead to relapse in certain types of leukaemia
What does genetic fingerprinting rely on?
The fact that eukaryotic DNA contains many repetitive non coding bases of DNA
What are non coding DNA bases known as?
Variable number tandem repeats (VNTRs)
How can VNTRs give a person’s genetic fingerprint?
Every person has a different number and length of VNTRs
What are the main stages of creating a genetic fingerprint?
Extraction Digestion Separation Hybridisation Development
What is Extraction in genetic fingerprinting?
The DNA is separated from the rest of the cell, and increased by PCR
What is Digestion in genetic fingerprinting?
The DNA is cut into fragments using restriction endonucleases, chosen for their ability to cut close to but not within the sequence
What is Separation in genetic fingerprinting?
Fragments of DNA are separated according to size by gel electrophoresis. Gel then immersed in alkali to separate double strand from single strands
What is Hybridisation in genetic fingerprinting?
Probes are used to bind with VNTRs, with their complementary base sequences. Carried out under specific conditions eg temperature, pH
What is Development in genetic fingerprinting?
X ray film put over nylon membrane, exposed to radiation and patterns appear of VNTRs
What are the uses of genetic fingerprinting?
Genetic relationships and variability
Forensic science
Medical diagnosis
Plant and animal breeding
How can genetic fingerprint be used for Genetic relationships and variability?
Can be used to resolve questions of paternity, as individuals inherit material from both mother and father
How can genetic fingerprinting be used for forensic science?
Suspects DNA can be compared to DNA from the crime scene
How can genetic fingerprinting be used for medical diagnosis?
Can be used to diagnose diseases such as Huntington’s, which results from a three base sequence at the end of a gene being repeated over and over again
How can genetic fingerprint be used for plant and animal breeding?
To prevent undesirable interbreeding during programmes in farms and zoos, and to identify desirable alleles
