Recombinant DNA Technology Flashcards
What is Recombinant DNA?
- Recombinant DNA molecules are DNA molecules formed by laboratory methods of genetic recombination to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome
Where are we with recombinant DNA technology so far?
- Therapeutic proteins (eg. Human growth hormone and human insulin) have been recombinantly produced in bacteria (E. coli)
- The first two recombinant proteins authorised for use in the USA as a drug therapeutic
- Many more therapeutics will be produced in bacteria in the future
Why is recombinant DNA important?
- Much of what we know about gene structure and function stems from molecular studies using recombinant DNA techniques
- The ability to clone, sequence and assess the function of genes is reliant on recombinant DNA techniques, and has aided in the construction and extended use of DNA and protein databases
Imagine you want to study the gene that is linked to cancer, or some other disease, what questions would you ask in preparation of finding the gene?
- What is the DNA sequence of the gene?
- What is the mutation(s) in the gene that causes disease?
- What is the function of the protein product of the gene?
- How is that function changed in the mutated protein?
- In which cells is the gene expressed?
- What are the promoter sequences that control the expression of the gene?
- Is the expression of the gene altered in cancer (or disease)?
The preservation of endangered species involves three major genetic solutions, what are they?
- Gene Banks
- Cloning and the Preservation of Endangered species
- Conservation Genetics
What are Gene Banks?
- Gene banks are helping to preserve endangered species by storing the biological and genetic material of animals that are under threat of extinction
What is the Frozen Ark Project?
- The Frozen Ark is an organisation that preserves endangered species
- The Frozen Ark Project strives towards a world where extinction rates are sustainable and not created by man. Where the beauty, splendour and practical solutions found in all species is noted and used by man for the good of mankind and for the good of all life on Earth.
What is Cloning and the Preservation of Endangered species
- Animal cloning is now a possibility and many species have been cloned. For conservation efforts this could mean that a clone is created from stored genetic material and implanted into the surrogate mother of a closely related species.
What is Conservation Genetics?
- Applies genetic methods to the conservation and restoration of biodiversity. Essentially the field combines several disciplines: molecular biology, ecology, population genetics, mathematical modelling, and evolutionary systems, with the aim of preserving biological diversity.
What is environmental DNA?
- Environmental DNA or eDNA is DNA that is collected from a variety of environmental samples such as soil, seawater, snow or even air rather than directly sampled from an individual organism
What are some examples of Environments where eDNA might be found and collected?
- Glaciers
- Permafrost/tundra
- Aquatic sediments
- Lakes and streams
- Terrestrial habitats
- Oceans
The first three are ancient environments while the latter three are modern
What types of environmental sampling is used to retrieve eDNA from the environment?
- Ice cores
- Soil/sediment core samples
- Freshwater/seawater samples
What DNA extraction procedures are used for getting the eDNA out of the environmental samples?
- The DNA extraction is carried out using procedures specific for the individual type of sample
What is PCR?
- Polymerase chain reaction (PCR) is a method widely used in molecular biology to make several copies of a specific DNA segment
What is the overall process of collecting eDNA?
- Find the environment (glaciers, tundra, etc)
- Gather environmental samplings (Ice cores, soil samples, etc)
- DNA extraction
- PCR amplification of extracted DNA
- Bioinformatic data processing
How is PCR amplification done?
- Using generic or species specific primers targeting biodiversity and subsequent sequencing of amplicons
- An amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events
What is Bioinformatic data processing
- Bioinformatics is a science field that is similar to but distinct from biological computation.
- Bioinformatics and computational biology involve the analysis of biological data, particularly DNA, RNA, and protein sequences.
Study the diagram of how eDNA is collected
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
How are Gene Coding and Recombinant DNA accomplished?
- Gene cloning is the isolation and amplification of a given gene
- A recombinant DNA molecule is a DNA molecule made by joining two or more different DNA molecules
- Both are accomplished using:
- Molecular scissors: Restriction Endonucleases
- Molecular glue: DNA Ligase
- As well as molecular glue and molecular scissors, we have the molecular equivalents of DNA production lines
What is Restriction Endonuclease?
- Naturally occurring enzyme present in many bacteria, also called restriction enzymes or RE
What is the function of Restriction Endnucleases?
- Natural functions can be likened to a bacterial immune system. Basic function of these enzymes is to cut up DNA. Any foreign DNA that enters the bacteria is cut up by the bacteria’s restriction endonucleases
- The bacteria’s own DNA is protected from digestion by the restriction enzymes, usually by a specific methylation pattern. Any infecting, foreign DNA is not methylated appropriately and so is digested
- Some restriction enzymes cut DNA in a random fashion, but others cut DNA strands at specific base sequences
What is Methylation?
- In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom by a methyl group
How is DNA protected from RE digestion via Methylation?
- Some restriction enzymes cut DNA in a random fashion, but others like EcoRI cut DNA strands at specific base sequences
- For example, The DNA strand of EcoRI will have a Methylated recognition sequence where the CH3 or some other methyl group will bond to the DNA strand. This prevents any enzyme binding which in turn prevents random DNA cutting.
Study the diagrams for The Protection of DNA from Restriction Enzyme Digestion by Methylation
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
How is Site specific RE used?
- Site specific restriction endonucleases are used in recombinant DNA technology
- RE recognise a specific DNA sequence, normally between four and eight base pairs long
- The first site-specific restriction endonuclease discovered was HincII (HindII)
How are site specific RE named?
- The first three letters of the name of the enzyme indicates the bacteria from which it was purified, e.g. HincII = Haemophilus influenzae
- If the enzyme is produced only in a particular strain, then a letter designating that strain is added (here c for strain Rc, d for strain Rd)
What does the restriction enzyme HincII (HindII) do?
- HincII (HindII) recognises the sequence “GTPyPuAC” and CAPuPyTG
- It cuts the double stranded DNA in the middle of the sequence and leaves two blunt DNA ends
How are sticky ends and blunt ends created?
- Different restriction enzymes cut the DNA in different ways
- The “ends” refer to the ends of the DNA strands
- Depending on where and how the RE cuts, it will produce either sticky or blunt ends
What are Sticky ends?
- Sticky ends on the other hand are generated by a staggered cut and they have unpaired bases or overhangs at the 3′ and 5′ regions. These overhangs are useful during the ligation as they ensure proper joining of the fragments
What are Blunt ends?
- Blunt ends are formed by a straight cut and they do not contain any unpaired bases or overhangs at the 3′ or 5′ regions. Hence, it is difficult to ligate the fragments with blunt ends.
Study the diagram of the cutting of DNA
- Google doc
What sequences are Restriction Endonuclease Recognition Sites usually found on?
- They are often palindromic sequences (Nucleotide pair sequences that read the same forward or backward from a central axis ‘*’ )
- E.g. EcoRI: GAATTC or CTTAAG
How long are the Restriction Endonuclease Recognition Sites usually?
- Usually either 6 bases long, but also 4 bases. A few restriction endonucleases recognise 8 base sequences (eg. NotI)
- Some restriction enzymes have the same recognition sequence, eg. SmaI and XmaI, but they make different cuts
- E.g. SmaI: CCCGGG, where the cut is downward and GGGCCC, where the cut is uppward (opposite strands)
- E.g. XmaI: CCCGGG, where the cut is downward and GGGCCC, where the cut is upward (opposite strands)
What are Isoschizomers?
- Restriction enzymes that recognise the same sequence
Study the table for the recognition sequences and cleavage sites of representative restriction Endonucleases
- Google doc
What is a “Digest” in genetics
- When fragments of DNA are cut by RE
What is the SV40 chromosome?
- Simian virus 40 (SV40) is a useful model system for numerous studies in the fields of eukaryotic gene transcription, DNA replication, and cell transformation.
- Thus, the viral genome contains promoters with complex arrays of regulatory elements as typically found in regulated cellular genes.
What are restriction maps?
- Restriction maps reflect true physical distances
- Restriction maps can be combined with other molecular techniques to construct physical maps of entire genomes
What are the steps for the Construction of Recombinant DNA Molecules In Vitro
- Step one = cut both of the DNA from 2 speices with an RE
- Step 2 = mix the cut (digested) DNA’s and incubate under annealing conditions
- Step 3 = Treat annealing DNA fragments with DNA Ligase
What is plasmid DNA?
- A plasmid is a small DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently.
How is Ligase used to make recombinant DNA?
- Ligase can join two pieces of DNA by forming the missing phosphodiester bonds between each strand of DNA
- Sticky ends created by restriction endonucleases (eg. EcoRI) facilitate this process
- Ligation takes place between 5’ -P and 3’ -OH termini
Study the diagrams for the Construction of Recombinant DNA Molecules In Vitro and DNA Ligase Function
- Google doc
