Lecture 2 Reading Flashcards
Brocks Chapter 11
1
Q
Brocks Chapter 11
A
Restriction enzymes
Electrophoresis (separation of nucliec acids)
Nucleic acid hybridization
Molecular cloning
2
Q
Restriction enzymes
A
- the original purpose of restriction enzymes is to degrade foreign DNA (eg. viruses)
- restriction endonucleases are in 3 types:
1. Type I and III: bind DNA at recognition sequences but cleave DNA further away
2. Type II: cleave within the recognition sequence - Type II is more commonly used for this reason
- cleavage sites are often palindromic
- most restriction enzymes are homodimers to ensure a double stranded break
- Type II enzymes most often make staggered cuts - ‘sticky ends’
- modification enzymes (methylases) protect host DNA from being degraded by restriction enzymes (eg. methylation of bases in the recognition sequence)
3
Q
Gel electrophoresis: separation of DNA molecules
A
- restriction enzymes produce fragments between 100-1000s base pairs long
- generates a restriction map of DNA, you can see the pattern of the cuts
- restriction analyses are used for: molecular cloning (which enzyme cuts which sites) and comparison of different but related DNA sequences (for classification of microbes)
4
Q
Nucleic acid hybridisation
A
- When DNA is denatured, the single strands can hybridise into double strands with other DNA/RNA strands that are (almost) complemetary
- used to detect, characterise and identify segments of DNA: finding related sequences in different chromosomes or to find the location of a specific gene
- nucleic acid probes are single strand segments that allow detection as they are labelled with fluorescent or radioactive labels
- Southern blotting - probes of known sequence are hybridized to target dna fragments that have been separated by gel electrophoresis
- hybridization is often used to identify genes after cloning
5
Q
Molecular cloning
A
- when a fragment of dna is isolated and replicated
- isolate desired gene
- move into a plasmid/virus or other vector
- as vector replicates, so does desired gene
- Isolation and fragmentation of source DNA
can be either: total genomic dna from organism (cut up with restriction enzymes), dna from an rna template, genes amplified by PCR, or totally synthetic dna made in vitro - Insertion of DNA into a cloning vector
vectors are small, independently replicating elements that carry and replicate cloned DNA segments. When the source DNA and the vector DNA are both cut with the same restriction enzymes it creates matching ‘sticky ends’ that help ligation - Introduction of the cloned DNA into a host
transformation is often used to get recombinant DNA into cells. Often results in a mixture of recombinants: DNA library (different clones can be purified from the mixture - shotgun cloning is often used in genomic analyses.)
6
Q
Selecting clones
A
- identification is usually through markers such as antibiotic resistance or plaques (with phage)
- also found by sequencing
- antibodies can be used to detect a protein of interest (protein acts as the antigen)
7
Q
Molecular Mutagenesis
A
- conventional mutagens introduce mutations at random
- in vitro mutagenesis (site-directed mutagenesis) uses synthetic DNA plus cloning techniques to introduce mutations into genes at precisely determined sites
1. Synthesise a short DNA primer containing the desired base change to allow pairing with ssDNA containing the target gene
2. DNA polymerase copies it into the rest of the gene
3. Inserted into host cell via transformation (selected for usually with an antibiotic resistance gene) - widely used vector is bacteriophage MI3: gives a dna molecule with a mismatch, after cell division only one of the daughter cells will carry the mutation, so are screened for
- Site directed mutagenesis can also be carried out using PCR:
short DNA molecule with mutation is the PCR primer. Mutation carrying primer is designed to anneal to the target in the middle with enough matching nucleotides on either side to be stable during PCR reaction. When the PCR amplifies the target DNA it incorporates mutations
8
Q
Applications of site directed mutagenesis
A
- used to investigate the activity of proteins with known aa substitutes
- gives detailed information to which aa are critical for enzyme structure and function
9
Q
Cassette mutagenesis
A
- when intervening DNA fragments are excised and synthetic fragments inserted (where one or more nucleotides have been changed) - called DNA cassettes
- Another type of cassette mutagenesis is called gene disruption
cassettes are inserted into the middle of a gene, disrupting the coding sequence. Cassettes used for making insertion mutations can be almost any size and can even carry an entire gene - when a cassette is inserted, cells gain AB resistance and lose the function of the gene where the cassette was inserted: knockout mutation
10
Q
Reporter genes
A
- reporter genes encode a protein that is easy to detect and assay
- used to report the presence or absence of a genetic element (eg plasmid), fused to other genes, fused to promotors of genes so that gene expression can be studied
- lacZ is a reporter that encodes enzyme b-galactosidase (that cleaves Xgal to produce blue colour)
- luciferase is a reporter that makes cells expressing it luminescent
- GFP is another reporter
11
Q
Gene fusions
A
- construct from two different genes
- if the promotor that controls a coding sequence is removed, the coding sequence can be fused to a different regulatory region (under the control of a different promotor)
- Two types of gene fusions:
- Operon fusions:
a coding sequence retains its own translational start site and signals fused to the transcriptional signals of another gene
assesses transcriptional regulation - Protein fusions:
two coding sequences fused so they share the same transcriptional and translational start sites and signals
assesses translational control
- Fusions are often used in studying gene regulation or to test the effects of regulatory genes
12
Q
Plasmids as vectors
A
- replication of plasmids in the host cell proceeds without chromosomal control (independent replication)
- plasmids are natural vectors because they carry genes that confer important properties for the host
- small size makes DNA easy to isolate and manipulate
- multiple copy number so many copies are present in each cell (high yields of DNA and high level expression of cloned genes)
- presence of selectable markers such as AB resistance genes
- vector transfer is mediated through electroporation or chemically mediated transformation
- pUC19 is a widely used vector: has a gene for ampicillin resistance and a lacZ gene that is a blue-white colour screening system and polylinker with multiple cut sites for different enzymes. Is a good vector:
- small (2686 bp)
- stably maintained in host E coli cell (50 copies per cell)
- amplified to high number by inhibiting protein synthesis with chloramphenicol
- polylinker has cut sites for a dozen enzymes
- ampicillin resistance marker
- detection of uptake seen by lacZ marker
13
Q
Cloning genes into plasmids
A
- insertional inactivation: plasmid and foreign DNA are cut with the same enzyme
- vector is linearized
- segments of foreign DNA are inserted into the open cut site and ligated into position with DNA ligase
- disrupts the gene
14
Q
Cloning hosts
A
- hosts should: be fast growing on cheap medium non pathogenic easy to transform genetically stable in culture have appropriate enzymes to allow replication of the vector
Prokaryotic hosts:
Ecoli commonly used, but can be problematic because it can potentially be pathogenic
lack of system to correctly modify eukaryotic proteins
gram negative outer membrane hinders protein secretion
B subtilis can be used instead but has plasmid instability
Eukaryotic hosts:
Saccharomyces cerevisiae most commonly used, good because they already contain RNA and post translational processing systems for production of eukaryotic proteins
Mammalian cells are expensive and difficult to produce large scale
Insect cell lines are simpler to grow, baculovirus is an insect viral vector
Plants can be cloned with tissue culture cell line or entire plant
15
Q
Transfection of Eukaryotic hosts
A
- introduction of DNA into mammalian cells
- mammalian cells can be transfected by adding DNA in combination with a variety of cationic carriers that bind to and protect DNA due to their positive charges
- in animal cells DNA can be taken up by:
1. Phagocytosis
2. Microinjection (DNA injected with a micropipette directly into the nucleus)
3. Electroporation is also used: pulsed fields in the presence of cloned DNA create small pores in the cytoplasmic membrane
- Microprojectile gun (gene gun): uses a propellant such as helium to fire DNA coated particles through a small steel cylinder at target cells, pierces the cell membrane but does not kill the cell
The gene gun can be used to get DNA into intact tissues, mitochondria and chloroplasts (not possible with electroporation)
