9: DNA based technologies Flashcards
genome definition
the complements of genes that an organism contains. In humans very little of the DNA actually encodes genes for proteins (~30,000)
genomics definition
the study of genes, how they are arranged, how many, what are they similar to
proteome definition
the complement of proteins that an organism produces from its genome and post translational modifications. It is dynamic and huge!
proteomics definition
study of the proteome, how these proteins are expressed, when they are expressed, what is their function, how do they interact
DNA cloning definition
to separate a gene from a larger piece of DNA (such as a chromosome) and replicate it
Basic concepts of DNA to protein central dogma. how does each step start and end?
replication: DNA polymerase starts at origin of replication sequence and ends at replication termination sequence
transcription: starts at transcription start site bracketed by 5’ sequence. different polymerases use different promotes. ends at transcription termination sequence
translation: starts at an AUG sequence that is bracketed by ribosome binding site. ends at a stop codon
Basic process of DNA cloning
- select a cloning vector: must contain all the necessary elements to convince host to amplify, transcribe, translate
- PCR: select your DNA of interest
- Ligate: join the pieces of DNA
- Transformation: transfer the recombinant DNA construct into a host cell (E coli)
- select the cells with harbor the recombinant DNA
types of vectors and size of insert they can carry
Plasmids: naturally occurring, circular pieces of double stranded DNA. Design to carry genes that are advantageous to host, can be autonomously replicated, and kept small and simple for use as cargo ship. Insert must be small, <3kb
Bacteriophage: linear double stranded DNA molecules that can be packaged into the head of a bacteriophage. about 1/3 of its genome is non-essential, can be replaced by foreign DNA of around 15kb. use when large amounts of lengthy DNA are needed
Cosmids: hybrid plasmids with lambda phage cos sequence (behave kind of like bacteriophage). somewhat unstable and sometimes difficult to maintain. Large insert possible, around 40kb
BACs/YACs: bacterial/yeast artificial chromosomes. for very large inserts, 300kb and YACs up to 3000kb
cloning plasmids all have:
- replicator (ori)
- selectable marker, eg antibiotic resistance gene
- cloning site or multiple cloning site (MCS)
what is the relationship between transformation efficiency and plasmid size
inversely proportional, smaller plasmids transform more efficiently. keep both the vector and insert as small as possible
What do you need to make a PCR work?
template
primers
DNA polumerase
dNTPs
describe the PCR process
heat strands to 98°C to separate (denature). add the primers and cool to anneal. add DNA polymerase to catalyze 5’-3’ DNA synthesis (only gos in 5 to 3!). repeat the process about 25 times to amplify 10^6 fold
what are restriction enzymes and their use?
In order to insert DNA you need to cut the plasmid and insert at a defined site which matches and can be ligated together. This is done by restriction enzymes (endonucleases) which recognize and cut double stranded DNA at specific restriction sites (a 4-6 bp palindrome). Native purpose to cleave foreign invading DNA. Can generate sticky or blunt ends
how to find restriction sites
in vectors: provided by companies that sell vectors, called multiple cloning sites (MCS)
in DNA with gene of interest: plug sequence into online search engine to find.
what if there are no convenient restriction sites around the gene of interest?
- linkers/adapters
- PCR to add a restriction site: add an overhang to the primer that encodes a restriction site sequence, so when the sequence is replicated in now contains a restriction site
What does ligase do?
seals the DNA backbone using ATP in the process
what are the options for combining restriction enzymes to get the most reliable result?
Cutting with a single RE is not super reliable, it can insert backwards. Cut with 2 REs to avoid this. most to least reliable:
- clone with two sticky ends
- clone with one sticky end and one blunt end
- clone with two different but compatible ends (doesn’t match but still anneals, destroys palindrome)
- clone with two blunt ends, last resort option. not very good
how to make competent (willing to accept exogenous material) cells for transformation
two ways:
- chemically: add CaCl2 and heat shock
- electroporation: apply high voltage
why use selection markers?
selection markers ensure that only the bacteria that take up the DNA grow. put an antibiotic resistance gene in the plasmid and grow in media containing antibiotics so only the bacteria harboring the plasmid can grow
special considerations for designing protein expression proteins
expression vectors: contain necessary elements for transcription and translation of genes
must insert the correct direction (use MCS)
bacterial promoter and operator sequences (upstream)
genes encoding regulators for promoter and operator
ribosomal binding site (upstream)
termination sequence (downstream)