Topic 8: Recombinant DNA technology Flashcards
What is meant by recombinant DNA technology?
The transfer of fragments of DNA from one organism, or species, to another.
Name three methods to produce DNA fragments:
1) Gene machine
2) Restriction enzymes
3) Reverse transcription/transcriptase
Describe how a gene machine is used and state the advantages of
using them to create DNA fragments.
DNA fragments can be created in a lab using a computerised machine.
Scientists first examine the protein of interest to identify the amino acid sequence, and from that work out what the mRNA and DNA sequence would be.
The DNA sequence is entered into the computer, which checks for biosafety, and biosecurity and that the DNA being created is safe and ethical to produce.
The computer can create small sections of overlapping single strands of nucleotides that make up the gene, called oligonucleotides.
The oligonucleotides can then be joined to create the DNA for the entire gene.
PCR is used to amplify the quantity and to make the DNA double-strand. This process is very quick, accurate and makes intron-free DNA so can be transcribed in prokaryotic cells.
Name and describe the process to make DNA fragments: Reverse transcription
Reverse transcription
This enzyme naturally occurs in viruses, such as HIV, and it makes DNA copies from mRNA.
A cell that naturally produces the protein of interest is selected.
These cells should have large amounts of mRNA for the protein.
The reverse transcriptase enzyme joins DNA nucleotides with complementary bases to the mRNA sequence. Single-stranded DNA is made (cDNA).
To make this DNA fragment double- stranded, the enzyme DNA polymerase is used.
Name and describe the process to make DNA fragments: Restriction enzymes
Restriction enzymes
Restriction endonucleases are enzymes that cut up DNA. They naturally occur in bacteria as a defence mechanism. There are many restriction enzymes that have an active site complementary in shape to a range of different DNA base sequences, described as recognition sequences, and therefore each enzyme cuts the DNA at a specific location.
DNA fragments can be amplified using PCR. Use the diagram and your knowledge to describe how.
The temperature is increased to 95oC to break hydrogen bonds and split the DNA into single strands.
The temperature is then decreased to 55oC so that primers can attach.
The enzyme DNA polymerase then attaches complementary free nucleotides and makes a new strand to align next to each template.
The temperature is increased to 72oC for this stage, the optimum for Taq DNA polymerase.
How do you prepare the DNA fragment for insertion?
Restriction endonuclease enzymes are used to cut out the gene (DNA fragment) of interest and cut at recognition sites leaving sticky ends.
The DNA fragments must be modified to ensure transcription of these genes can occur. A promoter region must be added at the start of the DNA fragment. This is a sequence of DNA that is the binding site for RNA polymerase to enable transcription to occur
A terminator region must be added – This is added at the end of the gene. It causes RNA polymerase to detach and stop transcription, so only one gene at a time is copied into mRNA.
Describe in vivo cloning
Restriction endonuclease enzymes are used to cut out the gene (DNA fragment) of interest and cut at recognition sites leaving sticky ends.
The isolated DNA needs to be joined to a vector, which will transport the DNA into the host cell.
Plasmids are commonly used vectors.
Restriction endonucleases are used to cut open the plasmid, the bacterial loop of DNA.
Using the same restriction endonuclease to create the DNA fragment and to cut open the plasmid ensures they have complementary sticky ends. DNA ligase (joins the sugar-phosphate backbone) is used to stick the DNA fragment in.
This is known as recombinant DNA now.
The recombinant plasmids are then introduced into the bacteria by transformation. Transformation involves mixing the plasmids and bacterial cells in a solution of calcium ions.
Sudden changes in the temperature of the solution make the bacterial membrane more permeable and the plasmid enters. (heat shock=42oC for 2 mins)
Not all the plasmids will enter the bacterial cell, and not all the plasmids will have taken up the DNA fragment, so the next step is to identify which bacterial cells did take up recombinant plasmids.
What is a gene probe?
Short, single-stranded pieces of DNA that are labelled radioactively or fluorescently so they can be identified.
What is an application of a gene probe?
To locate specific genes, e.g. for use in genetic counselling (identifying disease- causing genes) and personalised medicine.
What is DNA hybridisation?
When DNA is heated to separate the double helix into single strands, this is then mixed with complementary sequences of single-stranded DNA. Once cooled, the complementary strands will anneal.
What is personalised medicine?
Screening for the presence of particular alleles allows doctors to select medicines and health advice based on your genotype. Some drugs are more or less effective depending on the alleles you have, so this enables more effective and cost-effective treatment.
Describe each stage of the process of genetic fingerprinting
Collection & extraction
- The smallest sample of DNA can be collected for genetic fingerprinting. This could be from blood, body cells or hair follicles.
The DNA is extracted by cell fractionation and ultracentrifugation. If the sample of DNA is small, then PCR is used to amplify the amount of DNA.
Digestion
- Restriction endonucleases are added to cut the DNA into smaller fragments. Enzymes that cut close to the target VNTRs are added.
Separation
- The DNA samples are loaded into small wells in agar gel. The gel is placed in a buffer liquid with an electrical voltage applied. DNA is negatively charged, so the DNA samples move through the gel towards the positive end of the gel. An alkali is then added to separate the double strands of DNA.
Hybridisation
- DNA probes are short, single-stranded pieces of DNA complementary in base sequence to the VNTRs. The probes are radioactively or fluorescently labelled. Different DNA probes are mixed with the single- stranded DNA VNTRs on the agar gel for them to bind (hybridise). Development
The agar gel will shrink and crack as it dries, and therefore the VNTRs and DNA probes are transferred to a nylon sheet. The nylon sheet can then be exposed to x-rays to visualise the position of radioactive gene probes, or UV light if fluorescence probes were used.
Analysis
- The position of the DNA bands are compared to identify genetic relationships, the presences of a disease- causing gene and to match unknown samples from a crime scene.
What is the polymerase chain reaction?
method of copying or amplifying DNA fragments in a lab to provide a sufficient mass of DNA (e.g. for forensics or genetic fingerprinting)
What is needed for the polymerase chain reaction?
1- DNA fragment to be copied
2- TAG DNA polymerase which can join thousands of DNA nucleotides per minute. Obtained from bacteria in hot springs so they are thermostable
3- Primers- short, single stranded length of dna. They have a base sequence complementary to the start section of DNA to be amplified. Allows attachment of of DNA polymerase and prevents 2 DNA strands re-joining.
4- free DNA nucleotides
5- thermocycler that can regulate and vary temp over time
