Recombinant DNA Flashcards
tools used to make and study recombinant DNA (7)
- vectors
- cell cultures
- restriction endonuclease
- sequencing
- PCR
- site-direction mutagenesis
- CRISPR/Cas9
vectors (2)
- plasmids, bacteriophages, viruses
- a DNA molecule uses as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated/expressed
cell cultures
- used to grow larger amounts of DNA and to express gene products
PCR (2)
- used to amplify sequences of interest
- used to act as a diagnostic for the presence/absence of sequences
site-directed mutagenesis
- used to study the impact of sequence on gene function
CRISPR/Cas9
- used for gene editing and to study the function of genes
restriction endonucleases (2)
- a bacterial enzyme that defends against infection by phages
- recognize and cute at specific DNA sequences
how does bacteria protect itself against its restriction endonucleases
- bacterial DNA is typically methylated to protect its own sequences from its own restriction endonucleases
restriction enzymes (3)
- recognize short palindromic sequences (can be read the same both ways)
- generate sticky (asymmetrical cuts) or blunt (straight, even cuts) ends
- is exploited for cloning
recombinant DNA
- a molecule of DNA containing pieces of DNA from different species
DNA ligase
- enzyme that facilitates the joining of DNA strands together by catalyzing the formation of phosphodiester bonds
vector: bacterial plasmids (5)
- small circular extrachromosal elements
- replicate independently of the bacterial chromosome
- often carry genes for antibiotic resistance
- easily exchanged between bacteria
- many copies expressed per bacterial cell
vectors: viral vectors (3)
- viruses enter cells, replicate, and express their genes as part of their natural infection cycle
- viruses can be engineered to be and safe and to express cloned genes for study or therapy
- viral sequences may still be manipulated in plasmid form
what are the basic features a plasmid should contain (3)
- bacterial origin of replication (Ori)
- selectable marker (eg. antibiotic resistance)
- multiple cloning site (MCS)
additional features of plasmid (in addition to basic features) (4)
- general
- examples (3)
- variable and depend on plasmid’s ultimate use
examples: - blue-white selection for cloning genes
- strong promoters for high levels of expression
- sequences for insertion into plant or specific genomes
basic plasmid feature: bacterial origin of replication
- ability to replicate independently of the bacterial chromosome
basic plasmid feature: selectable marker
- easier to select and isolate transformed cells
basic plasmid feature: multiple cloning site
- contains unique restriction sites for ease of cutting and pasting DNA sequences into plasmids
higher copy plasmids (2)
- definition
- advantage
- more copies of the plasmid per cell
- more material to work with and many more copies of desired DNA product per cell
what are some applications of plasmids? (4)
- ease of selection for successfully cloned sequences
- promoter traps/enhancer traps
- manipulate genes in bacteria for expression in yeast
- create transgenic plants
PCR components (4)
- DNA template with sequence of interest
- dNTPs
- primers
- polymerase
PCR basic steps (4)
- denaturation
- annealing (of primers)
- extension (with dNTPs by polymerase)
- repeat cycles
why do we need to”cut” and “paste” DNA pieces together? (4)
- for study of genes and genetic elements
- to engineer organisms for study/use
- mass production of useful biological molecules
- for gene therapy
molecular cloning (2)
- involves a collection of methods that allow DNA to be inserted into vectors that can be grown in bacteria
- an alternative to PCR
what do we do with cloned DNA (2)
- can be manipulated for study
- for production
advantages of molecular cloning
- does not require knowledge of the sequence to be cloned; each clone generates a different sequence
disadvantages of molecular cloning (2)
- need to generate a gDNA or cDNA library
- need a good method to identify the gene of interest; need a good screening method
what are the advantages of PCR (2)
- ability to amplify only the gene of interest for cloning
- PCR can also be used to screen for clones of interest
disadvantages of PCR
- need to KNOW THE SEQUENCE in order to design the PCR primers
insulin
- hormone secreted by the pancreas to regulate glucose uptake from the blood (blood sugar level)
type 1 diabetes
- failure to produce insulin (through mutation or destruction of pancreas cells that produce insulin)
- now controlled by insulin injections
insulin production before recombinant DNA cloning (2)
- need for 56 million animals per year to have enough animal pancreases producing insulin for USA demand
- risk of developing allergic reaction to animal insulin as it is different from human insulin
insulin production after recombinant DNA cloning
- expressed cDNAs for both A and B light chains in E. coli, then chemically liked them to form fully processed insulin
steps in cloning human insulin gene
- cut open vector with restriction endonuclease
- ligate cDNA for human insulin into restriction site
- introduce into cultured cells
- screen for the clone with the sequence of interest and culture that clone
- purify human insulin from cells (E. coli) or from solution (from yeast cells)
molecular cloning OVERVIEW (3)
- definition
- advantage
- disadvantage
- the isolation and insertion of DNA sequences of interest into a vector so that the sequences are easily replicated and manipulated
- does not require previous knowledge of the sequence of interest
- may require more work to identify the clone of interest
