What are primers and probes?
Short base-pairs sequences of either DNA or RNA, designed to attach to a single stranded base pair sequence. The longer the sequence, the more specific. Vital in a wide range of microbiological tools, e.g GE, FISH, PCR, cloning, sequencing etc
Primers: A strand of nucleic acids that serves as a starting point for DNA replication, it is necessary as DNA polymerase enzyme cannot start by itself - it can only add to an existing strand on the DNA.
- Starts at the 3’ end and copies the opposite strand
- Used to sequence and amplify DNA
Probes: Designed to attach to either RNA or DNA
Main differences from a primer:
- Applied to whole cells, so no need to extract the nucleic acids from the cell first
- Designed only to bind
- Have a fluorochrome attached to allow detection
DNA regions
- Generic (conserved) base-pair sequence: a sequence that is present in for example all eubacteria
- Specific base-pair sequence: is only present in for example a specific genus or a specific species
What is PCR and its its applications?
Polymerase chain reaction Allows amplification of DNA, it can be rapidly cloned in a test tube instead of in a cell Applications: - Basic molecular biology research - Generates DNA for cloning/blotting probes - Clinical testing - Forensic science - Bioremediation, wastewater..
What is taq polymerase?
A thermo stable polymerase enzyme used when DNA needs to be heated up for separation
PCR requirements
- Thermocycler (allows rapid heating and cooling)
- Template DNA to be amplified
- Two primers (complementary to the 5’ and 3’ region respectively)
- Deoxynucleosidetriphosphates (dNTPs) (building blocks for new DNA strands)
- Buffer (reaction media)
PCR Steps
- Denaturation: Heat (near 100) melts the DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
- Annealing: temp cooled, annealing of the primers to the single stranded DNA template. The annealing temp is 3-5 degrees C below the Tm of the primers used. Stable DNA-RNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
- Extension: T=Toptimum polymerase enzyme used, slightly heated again. Synthesises a new DNA strand complementary to the DNA template strand
What can you do after PCR?
- Use general probes which will amplify all the DNA
- Amplify a gene from a person
- Amplify a colony of bacteria
- Amplify a specific region to generate probes or selected genes
What is gel electrophoresis?
Technique that allows for separation fo DNA, RNA, or proteins. The DNA/RNA is extracted out of the cell first.
Consists of a box containing a gel (usually a cross-linked polymer). An electric current is passed through the gel to move the molecules. Separation is based on how fast sequences migrate through the gel, the DNA fragments are negatively charged so they move to the positive electrode. Usually, all the fragments have the same amount of charge per mass so small fragments tend to move through the gel faster. Each separate sequence can then be removed and analysed afterwards or compared to standard gels.
Industrial biological wastewater treatment problems
Hard to degrade compounds require specialised bacteria (chlorobenzene, dichloroethane etc which are often uses as solvents in industry)
Common scenario: batch processes -> change in the production line -> changes in the wastewater composition -> no bacteria capable of degrading the specific compound present
What is FISH used for and what are its main advantages?
- Used to localize the presence/absence of a target DNA/RNA sequence in whole cells
- Main advantages:
Doesn’t require extraction of DNA, no need to cultivate (immediate results), spatial location of cells or genes.
Applied to identifying bacteria/yeast/cells of interest, or detect where on the chromosome a specific DNA sequence is located
What is the purpose of PCR?
To make many copies of an organism’s DNA sequence so a small number of organisms will become large enough to be identified
Where does taq polymerase start copying at?
RNA primers attached to the end of the desired genes
What enzymes catalyse the formation of covalent bonds that hold together the sugar-phosphate backbone of the DNA molecule?
Ligases
In what direction does replication proceed in?
A 5’ to 3’ direction
Considering DNA replication along the 5’ to 3’ template strand, what event takes place first?
Primase adds an RNA polymer
The number of amplified pieces of DNA equal what after 5 cycles of PCR?
32
Is it possible to use PCR to produce many copies of all the DNA of one chromosome?
No, PCR copies short sequences only
Does PCR require knowledge about parts of a target DNA sequence to be amplified?
Yes
