Biotechnology
The industry has grown up around the use of the three-dimensional structure of DNA
Molecular Genetic Techniques
(Extra-for fun-flip the card!)
Molecular genetic techniques are used to locate, analyze, alter, sequence, study, and recombine DNA sequence
Key Innovations Include
1) The development of recombinant DNA technology
2) The invention of the polymerase chain reaction
3) The development of quick and accurate methods of determining DNA sequences
4) The engineering of CRISPR-Cas systems for accurate and efficient editing of genome sequence
Ex. Molecular Genetical Crops
Recombinant DNA technology
is a set of molecular techniques on isolating,
Gel Electrophoresis
Is a standard technique for separating molecules on the basis of their size and electrical charge
(The porous gel made from agarose)
Small DNA fragments migrate more than larger DNA fragments
What are the steps of gel electrophoresis?
- DNA samples containing fragments of different sizes are placed in well in an agarose gel
- An electrical current is passed through the gel
- All DNA fragments move towards the positive pole; small fragments migrate faster than larger fragments
(After electrophoresis, fragments of different sizes have migrated different distances) - A dye specifies for nucleic acids is added to the gel
- DNA fragments appear as bands on the gel
What would happen if DNA fragments are still too small to see? (How would the problem be addressed)
The DNA fragments are still too small to see, so the problem of visualizing them must be addressed
Visualization can be accomplished in several ways…
***The simplest procedure is to stain the gel with a dye specific for nucleic acids, such as ethidium bromide
***DNA fragments can be visualized by adding a label to the DNA before it is placed in the gel – chemical labels can be detected by adding antibodies or other substances that carry a dye and will attach to the relevant DNA, which can then be visualized directly
What is a probe?
A DNA or an RNA molecule with a base sequence complementary to a sequence in the gene of interest
How to amplify a DNA?
(Magnify or make more of the DNA)
There are two basic approaches to amplifying a specific DNA fragment: replicating the DNA within cells – in vivo – and replicating the DNA enzymatically outside of the cell – in vitro
Gene cloning
An amplification technique
- The process of inserting the DNA into bacteria
- selecting and growing the bacterial cells that have incorporated it
- The bacteria cells are then lysed to release their DNA
- Desired fragment is isolated from the rest of the bacterial DNA
- Identical copies – clones – of the original piece of DNA are replicated within bacterial cells
What is a disadvantage with gene cloning?
A major disadvantage include…
1) Time required for the process
2) Labour-intensive
3) Require a large number of steps that are difficult to automate
What is the advantage with gene cloning?
Typically, gene cloning copies DNA with great accuracy
Polymerase Chain Reaction – PCR
It uses a DNA polymerase to continually replicate the DNA
What are steps of PCR?
- DNA is heated to 90-100 degrees celsius to separate the two strands
- The DNA is quickly cooled to 30-65 degrees celsius to allow short single-stranded primers to anneal to their complementary sequences
- The solution is heated to 72 degrees celsius; DNA polymerase synthesizes new DNA strands creating two new double-stranded DNA molecules
- The entire cycle is repeated – each time the cycle is repeated – the amount of target DNA doubles
Tax Polymerase
This enzyme found in bacteria/Archaea that live in the hot springs of yellow national park are used in PCR because it is a high temperature process
Reverse Transcription PCR
PCR technique used to amplify RNA
- First convert RNA into complementary DNA using reverse transcriptase
- The complementary DNA can be amplified like RNA
Limitations of PCR
- The use of PCR requires prior knowledge of at least part the sequence of the target DNA so that the primers can be constructed – we need to know a little pit of the coding gene to make the corresponding primers
- Contamination – laboratory techniques must be highly controlled
- Accuracy – Tax polymerase does not have the capacity to proofread like DNA polymerase
- The size of the PCR that can be amplified by standard Taq polymerase is usually less than 2000 bp
Cloning Vector
:a stable, replacing DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell
What three important characteristics does an effective cloning vector have?
- an origin of replication, which ensures that the vector is replicated within the cells
- selectable markers, which enable any cell containing the vectors to be selected or identified
- one or more restriction sites into which a DNA fragment can be inserted into
Plasmid
Circular DNA molecules that exist naturally in bacteria – are commonly used as vectors for cloning DNA fragments in bacteria
What are steps of creating recombinant DNA with plasmids?
- First, a cloning vector must contain an origin of replication recognized in the host cell so that it is replicated along with the DNA that it carries
- Second, it should carry selectable markers – traits that enable cells containing the vector to be selected or identified for
- Third, a cloning vector needs a single cleavage site for each of one or more restriction enzymes used
