Outcome 1: DNA, Proteins, CRISPR, Plasmids, PCR, GMO's Flashcards
What type of bonding is between the nitrogenous bases?
Hydrogen bonding
What type of bonding is between the deoxyribose sugar and the phosphate group?
Phosphodiester bond
What will a 5’ end of a DNA strand end with?
Phosphate group
What will a 3’ end of a DNA strand end with?
Deoxyribose sugar
Which of the nitrogenous bases are purines?
A and G
Which of the nitrogenous bases are pyrimidines?
C, U and T
How many hydrogen bonds do A and T have?
2
How many hydrogen bonds do C and G have?
3
When nucleotides are joined what is the byproduct and what type of reaction occurs?
Water, condensation reaction
What is semi conservative replication?
Replication where one strand is the original DNA and one is new.
What are the four differences between RNA and DNA?
- RNA contains ribose instead of deoxyribose.
- RNA replaces Thymine with Uracil.
- RNA is single stranded
- RNA are much shorter and less stable than DNA
Role of messenger RNA
(mRNA)
Carries DNA code to ribosome for protein synthesis.
Role of transfer RNA
(tRNA)
Delivers amino acids to ribosome for protein synthesis by matching a anticodon to a codon.
Role of ribosomal RNA
(rRNA)
Component of the ribosome that associates connections for protein synthesis.
Where does transcription occur?
Nucleus
Where does translation occur?
Cytoplasm (outside the nucleus in eukaryotes)
What three ways is pre-mRNA modified?
- Methyl cap added to 5’ end
- Introns removed
- Poly-A-tail added to 3’ end
Which end can nucleotides only be added to?
3’
Order of non template strand
5’ to 3’
Order of template strand
3’ to 5’
Order of pre-mRNA
5’ to 3’
Regulatory gene
Section at the beginning of a prokaryotic gene.
Structural gene
Section of the gene that codes for a protein.
Operon
Series of genes under control of one promoter
Which type of organism has introns and exons?
Eukaryotes only
Intron
Non-coding section of DNA that is edited out.
Exon
Coding section of DNA that is kept in the final product.
What binds to the promoter region of DNA
RNA polymerase
What are the five genes controlled by the trp operon?
trp E, trp D, trp C, trp B, trp A
How does the trp repressor bind and unbind to the operator?
If there is two tryptophan available to bind to the repressor, then it changes shape and attaches to the operator.
Purpose of the trp repressor?
To bind to the operator when there is an excess of tryptophan and block RNA polymerase
What are the polymers proteins are made of called?
Polypeptides
What are monomers that made up proteins called?
Amino acids
If a word ends in “in” what is it likely to be?
A protien
If a word ends in “lyse” what is it likely to be?
An enzyme, type of protein
What is a proteome?
Whole set of proteins produced by a cell
What parts of the molecular structure of amino acids is always the same?
- Amine group
- Carboxylic acid group
- Central carbon atom and attached hydrogen.
Amino acids can be… hydr- or hydr-
Hydrophobic or hydrophilic
What part of the molecular structure of amino acids is different?
The R group
What is the primary structure of protein?
A linear sequence of amino acids
What is the secondary structure of protein?
- and what 3 things can it form?
Boiled and folded portions form (formation of: alpha helix, random loops, beta pleated sheet)
Can a secondary structure of a protein function?
No
What is the tertiary structure of a protein?
Proteins form into functional shapes
What is the quaternary structure of a protein?
2 or more polypeptides join to more a mature protein
What type of bond is between each amino acid?
Peptide bond
Where are proteins made?
Rough endoplasmic reticulum
Where is the lumen located?
Inside the rough endoplasmic reticulum
What happens to proteins inside the lumen?
Polypeptides are folded and packaged into vesicles and transported to golgi aparatus
What happens to proteins in the golgi aparatus?
Further processing in cisternae and labelled to leave the cell
What are the types of genetic engineering?
- Knock out
- Knock in
- Transgenic
What is genetic engineering?
Changing the genetic sequence of an organism
What is a transgentic organism?
Where DNA from one organism is inserted into another
3 important rules around gene modification
- specific location is important for strength
- No disrupting genes
- If done randomly will have negative consequences
How is a knock out organism produced?
Produced by cutting out gene segments to prevent expression
How is a knock in organism produced?
Produced by inserting genes into specific locus that is controlled by a specific promoter
All ___ are ___ ,but not all ___ are ___
All TGO’s are GMO’s, but not all GMO’s are TGO’s
Restriction enzymes can also be called
Restriction endonucleases
What is a restriction fragments?
DNA molecules cut into smaller pieces
What is this?
G ACGTC
CTGCA G
Sticky ends
What is this?
CC GG
GG CC
Blunt ends
What is a restriction site?
Specific sequence of DNA that enzyme will cut
What is the only enzyme that sticks DNA back together?
DNA ligase
What is a recombinant plasmid?
rejoined/edited DNA from a bacterial plasmid
What does DNA polymerase produce?
Exact copies of DNA
What does PCR stand for?
Polymerase chain reaction
What is Ligase?
The only enzyme that joins DNA
How many cycles does a PCR do?
30
What machinery is used to perform PCR?
A thermal cycler
What are the 3 steps of PCR?
- Denaturation
- Annealing
- Elongation
What occurs during denaturation?
DNA is separated into single strands (~90C, 1 minute)
What occurs during annealing?
DNA primers attach to 3’ ends of target DNA (~55C, 1 minute)
What occurs during elongation?
Heat tolerant DNA polymerase binds to primer to copy strand (~72C, 2 minutes)
What is gel electrophoresis?
The separation of molecules by size
What charge does DNA have?
Negative charge
What charged end is DNA placed at in gel electrophoresis?
The negative end
What is the ladder in a gel electrophoresis?
A set of known values which create a scale to identify unknowns
What is a polymorphisms?
Regions of DNA that show high variablility
What percentage of DNA is non-coding?
99%
What is a short tandem repeat (STR)?
non-coding regions of repeating elements in DNA
What does this mean [GAAT]5
GAAT repeated 5 times
What is the purpose of a PCR reaction?
Generate copies of STR regions for analysis
What is an electropherogram?
The results produced from a persons DNA profile
What is CRISPR cas9?
Genome editing system, based on bacterial defense
What does CRISPR stand for?
Clustered
Regularly
Inter
Spaced
Palindromic
Repeats
What does palindromic mean?
Read the same way on both sides e.g.
AAATTT
TTTAAA
What is target DNA (CRISPR)?
The DNA inserted by the bacteriophage to be scanned by cas 1 and 2
What is crRNA?
RNA section that matches the virus and has a target RNA added
What is tracer RNA?
A section of RNA that attaches to the repeats and acts an anchor for the gRNA
What is crRNA called inside the cas 9
gRNA
What does cas 1 and 2 do when they locate PAM?
Cut a section roughly 20-26 base pairs upstream of the PAM
What are the virus DNA’s called in the CRISPR array?
spacers
What is between each spacer in the CRISPR array and what is it made up of?
Repeats, made up of palidromes
What does PAM stand for?
Protospacer adjacent motif
What is the section cut from the virus DNA called before its added to the CRISPR array?
Protospacer
What is the CRISPR array?
Chronological record of previous viral infections
What is pre-crRNA cut with?
RNAase
How is pre-crRNA modified to become cr-RNA?
Tracer RNA’s attach to the repeats and the sections are cut up
What does cr-RNA bind to?
Cas9 Protein
What is called when crRNA binds to Cas9 Protein?
CRISPR-Cas9 complex
What is crRNA called when binded to cas9?
gRNA or guideRNA
What does the CRISPR Cas9 complex do?
Scans DNA for PAM’s
What does the CRISPR cas9 complex do with a PAM?
Unzips DNA to check if template is complementary, if it is then the cas9 will make a double stranded break roughly 4 bases upstream from the PAM
What is the difference between gRNA and sgRNA
guide RNA is naturally occurring, single guide RNA is made in a lab
Describe non homologus end joining
Proteins 2 attach to ends and then attract proteins 1, then they attract proteins 3 which form a spring to pull DNA back together, then DNA ligase joins DNA by repairing sugar phosphate bond
What is non homologus end joining used for? By scientists
to knock out genes
Why is homologous end joining preferred over non homologous end joining?
Homologous end joining is more accurate but requires a spare piece of homologous DNA
What are the steps in homologous end joining?
- 5’ ends are cut back to create overhang
- Strand is take down to homologous pair to be repaired with DNA polymerase
- Strand reattaches and other side is repaired
- DNA Ligase connects sugar phosphate bonds
What is a vector?
Vehicle that is used to deliver genetic material to a target cell via horizontal gene transfer
What is non-viral delivery?
commonly used bacterial plasmid (circular DNA)
What is viral delivery?
Insertion of a viral vector into a cell via the use of bacteriophages
What are the risks of viral delivery?
random insertion into genomes could affect key host genes
What is insulin?
Hormone that promotes uptake of glucose by cells
What is type 1 diabetes?
Insulin producing cells in pancreas are attacked by immune system
What is type 2 diabetes?
Insulin receptors no longer accept insulin
Outline the 7 steps in making human insulin using bacterial plasmids
- Restriction enzyme cuts section of plasmid
- Isolate gene of interest (remove introns)
- Make recombinant plasmid - antibiotic resistance gene added (reporter gene)
- Genetic transformation (taking up the plasmid
- Bacterial reproduction
- Identify bacteria that have transformed using reporter genes
- Extract insulin and purify
What is the structure of insulin?
made up of 2PP chains (quaternary structure)
What is reverse transcriptase?
reverses transcription (RNA->DNA)
What are the A and B chains in relation to insulin?
The two chains are joined together after creation to form insulins quaternary structure and disulfide bonds
What is B-galactosidase?
added as a reporter gene, turns bacteria blue in the presence of lactose
