L2: Methods in Molecular biology Flashcards
Gel electrophoresis
Laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size.
In what ways can DNA be manipulated?
By restriction digestion and PCR
Restriction digestion
DNA is cut at specific sites, dictated by surrounding DNA sequences.
PCR standard reaction
- Water
- Buffer (cations and pH)
- DNA to amplify (template)
- Enzyme (eventually Taq polymerase)
- Two primers
-dNTPs
PCR (polymerase chain reaction)
Allows for rapid amplification of a specific segment of DNA in vitro. PCR makes billions of copies of a specific DNA fragment or gene, which allows the detection and identification of gene sequences.
Restriction enzyme cloning
Uses DNA restriction enzymes to cut a vector and insert it at specific locations so that they can easily be joined together by DNA ligase to create recombinant DNA.
Gateway cloning
Allows transfer of DNA fragments between different cloning vectors while maintaining the reading frame. It has replaced the use of restriction endonucleases.
Gibson assembly
Molecular cloning method allows for the joining of multiple DNA fragments in a single, isothermal reaction
TA cloning
One of the simplest and most efficient, methods for cloning PCR products. Exploits terminal transferase activity of certain thermophilic DNA polymerases, including TAQ polymerase.
E. coli has many uses
Produces plasmids + proteins, host to phage P1(Cre recombinase, enzyme), phage lambda (vectors..), phage T4 (DNA pol ligase), and enzymes (DNA pol, Klenow fragment, EcoRI)
How does E.coli grow?
Grows fastest between 37-38°C. Likes to eat: peptone-trypsin digested casein, yeast extract, and NaCl. Easy to grow in the lab (oxygen levels can be low or atmospheric)
What are restriction endonucleases?
Enzyme that cleaves DNA into fragments at or near specific recognition sites along the molecule. Enzymes type I and II and bacterial defense mechanism against phage.
Enzymes type I
Cleaved DNA with specific sequences (protects its own DNA via methylation)
Enzymes type II
Most are palindromic (same sequence on both strands). Digest in the site, at the same position of each strand.
What is meant by “sticky ends”?
Cutting of DNA molecules (from restriction endonucleases) results in the formation of either sticky ends or blunt ends of DNA, depending on the restriction enzyme endonucleases you use.
What is meant by “sticky ends”?
Cutting of DNA molecules (from restriction endonucleases) results in the formation of either sticky ends or blunt ends of DNA, depending on the restriction enzyme endonucleases you use.
Plasmid
Small extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can be replicated randomly.
Plasmid DNA’s
Typically come from E.coli. Are circular: easy to amplify in cells and in test tubes. Coiled: easy to separate from genomic DNA. Purify with alkali denaturing lysis. Needs to be recognized by the cell’s machinery (origin of replication). Needs to be beneficial to the cell (drug resistance).
Plasmid: pBSKSII
Gel electrophoresis
Provides a solid substrate to separate biomolecules in solution. Analyze reactions or natural samples. Purify specific fragments.
Sanger sequencing
Method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides. It is based on DNA polymerase rxns.
Standard reaction for Sanger sequencing
- Water
- Buffer
-DNA to sequence - Enzyme
- DNA primer
- Label-radioactive or fluorescent
- dNTPs
- ddNTPs
Site-directed mutagenesis
Method used to create specific, targeted changes in double-stranded plasmid DNA. Primers carry modifications, incorporated into amplified DNA.
Cloning (DNA tech)
- Isolating DNA from a natural source and replicating it in vitro (E.coli)
- Mutating a plasmid DNA and making lots of identical copies.
Cloning: Capturing genes
Plasmid library–> collection of plasmid clones that contain random genome fragments from a test species.
BAC (bacterial artificial library)–> Like a plasmid but holds 300kb. Requires a sequence for replication, maintenance and partitioning.
Colony hybridization
A method used to identify a colony containing a region of interest. Colony hybridization is a lb technique used to identify specific DNA sequences within a mixed population of microorganisms.
Cloning- mutating plasmids
Specifically designed to introduce random mutations into the DNA they carry, allowing to study of the effects of mutations on gene function. The mutations can be introduced by chemical or physical treatments, or by using error-prone DNA polymerase. Mutated plasmids can then introduce into cells where they replicate and produce a population of cells with different mutant versions of the target gene.
Plasmid DNA
- Open with restriction endonucleases
- B/W screen
- High copy number
- Highly accurate
-Digest with EcoRI
EcoRI (R.E.)
Restriction endonuclease (cleaves DNA at restriction sites). Firstly, isolated from E.coli. The recognition site is 5’-GAATTC-3’, and the enzyme cleaves DNA molecules between G and A residues, producing 5’-phosphate and 3’-hydroxyl groups on the ends of the cleaved DNA. Ends can be ligated to other DNA molecules allowing for the insertion of new DNA sequences into existing plasmids or for joining DNA fragments.
Apal
TA cloning
Cloning method (DNA fragments into plasmids) that uses terminal transferase activity of Taq polymerase. Taq polymerase adds a single dNTP to the 3’ end of a DNA molecule creating a small extension. Firstly the DNA fragment is amplified by PCR, and then the 3’ ends of amplified DNA are treated with Taq polymerase and ligated to a plasmid vector (has been cut by R.E.s).
Gateway cloning system
-Directional cloning
- maintains a reading frame
- No restriction enzymes
- No ligation
- 1 hour, room temp. reaction with 99% efficiency
- No re-sequencing
- Compatible with automation
- Reversible reactions
