19. Genetic Technology Flashcards
(51 cards)
Define the term recombinant DNA
DNA made by artificially joining together pieces of DNA from two or more different species
Why are fluorescent proteins being used as markers more often than antibiotic resistance?
Why fluorescent?
-They are easier to identify
-More economical
-No risk of antibiotic resistance
Why not antibiotic resistant genes?
-There is a risk that the antibiotic resistant genes could be accidentally transferred to other bacteria including pathogenic strains creating pathogenic antibiotic resistant bacteria
-If the resistance spreads to other bacteria this could make antibiotics less effective
What are the basic steps in producing a GMO?
- Gene is identified and is cut from a chromosome made from mRNA by a reverse transcriptase or synthesized from nucleotides.
- Multiple copies of the gene are made using the technique called the Polymerase chain reaction
- The gene is inserted into a vector which delivers the gene to the cells of the organism
- The vector takes the gene into the cells
- The cells that have the new genes are identified, often by using marker genes and cloned.
Describe 3 ways we can obtain genes for genetic engineering
- Use restriction endonucleases to cut the DNA slightly above and alrighty below the gene
- Get mRNA without introns, use reverse transcriptase to produce cDNA, use DNA polymerase to make into a double stranded DNA
- If you know the sequence of the gene, feed into a DNA synthesizer machine and artificially synthesize the gene.
Describe the formation of recombinant human insulin
mRNA is extracted from pancreatic B cells and it is used as a template for reverse transcriptase to make single stranded DNA. It is then used as a template for DNA polymerase to make double stranded DNA. Plasmids from the bacteria are cut with same restriction enzyme as that used to cut the cDNA. The ligase enzyme joins the DNA sugar-phosphate backbone to produce a recombinant plasmid. The recombinant plasmid is introduced into bacteria. Bacteria grow in a suitable medium and replicate the plasmids to produce many GM bacteria.
Why are antibiotic- resistance genes not used as markers anymore?
Because of the risk of creating pathogenic antibiotic resistant bacteria
How can you identify which bacteria have taken up a specific plasmid?
The gene for the enzyme is inserted into plasmids- this enzyme makes a protein called GFP (green fluorescent protein) and when ultraviolet light is shone onto it the one that glow green are the genetically modified ones.
What controls the expression of genes?
A promoter
What is a promoter?
A length of DNA that includes the binding site for RNA polymerase where transcription of a gene begins. It allows RNA polymerase to bind to DNA and also ensures that it recognizes which of the two DNA strands is the template strand.
What is gene editing?
A form of genetic engineering in which the genome of an organism can be changed by deleting, inserting or replacing a length of DNA using a method such as CRISPR/Cas9 system
Explain the advantage of using CRISPR/cas9 over using restriction enzymes
They can be used to cut, insert or delete or modify genes at precise locations
Describe how CRISPR/cas9 can be used to cut a DNA strand
introduce Cas9 and gRNA. The gRNA will attach to the target sequence and Cas9 will cut the strand
What is PCR - polymerase chain reaction?
An automated process that amplifies selected regions of DNA using alternate stages of polynucleotide separation and DNA synthesis catalyzed by DNA polymerase
What are the three stages of the PCR?
Denaturation
Annealing
Elongation
Describe what happens in the first step of the PCR?
Denaturation.
The DNA is heated to 95 degrees Celsius
This breaks the hydrogen bonds and separates the double stranded DNA
Describe what happens in the second stage of the PCR reaction
Annealing.
Primers are added on either side of the length of DNA which is being amplified (H bonds) they are required because DNA polymerase cannot begin synthesizing DNA without an existing strand to build on. This requires a temperature of 60 degrees Celsius
Describe the third stage of the PCR reaction
Elongation/ Extension.
DNA polymerase then uses the dNTPs or the free DNA nucleotides to build new strands of DNA against the exposed ones at a temperature of 72 degrees Celsius. The formation of the new strands uses TAQ polymerase
Why do we use TAQ polymerase in PCR?
It is not destroyed by the Denaturation stage so it does not have to be replaced during each cycle.
It’s high optimum temperature means that the temperature for the elongation stage does not have to be dropped below that of the annealing stage
Explain the difference between a primer and a probe
- A primer is a short strand of DNA or RNA that provides a starting point for DNA synthesis during PCR
- A probe is a short single stranded piece of DNA or RNA that is labeled and binds to a specific DNA sequence to detect the presence of that sequence
What is gel electrophoresis?
A technique that is used to separate charges molecules, by differential movement, through a gel in an electric field.
What are the 3 factors that determine how a charged molecule moves within the gel
-net charge (negatively charged molecules move towards anode, positively charged molecules move towards the cathode)
-size- smaller molecules move faster
-composition of the gel- the size of the pores within the gel determines the speed with which fragments of DNA move
What are the two types of gels used in gel electrophoresis and why?
- Polyacrylamide gel- used for separating small fragments
- Agarose gel- used for separating fragments that are between 100 base pairs and 50 000 base pairs in length
Why is a buffer solution used in gel electrophoresis?
Maintains ph and allows electric current to flow
Which electrode does DNA move towards?
The anode because DNA is negatively charged
