Recombinant DNA Flashcards
(35 cards)
1
Q
tools used to make and study recombinant DNA (7)
A
- vectors
- cell cultures
- restriction endonuclease
- sequencing
- PCR
- site-direction mutagenesis
- CRISPR/Cas9
2
Q
vectors (2)
A
- 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
3
Q
cell cultures
A
- used to grow larger amounts of DNA and to express gene products
4
Q
PCR (2)
A
- used to amplify sequences of interest
- used to act as a diagnostic for the presence/absence of sequences
5
Q
site-directed mutagenesis
A
- used to study the impact of sequence on gene function
6
Q
CRISPR/Cas9
A
- used for gene editing and to study the function of genes
7
Q
restriction endonucleases (2)
A
- a bacterial enzyme that defends against infection by phages
- recognize and cute at specific DNA sequences
8
Q
how does bacteria protect itself against its restriction endonucleases
A
- bacterial DNA is typically methylated to protect its own sequences from its own restriction endonucleases
9
Q
restriction enzymes (3)
A
- 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
10
Q
recombinant DNA
A
- a molecule of DNA containing pieces of DNA from different species
11
Q
DNA ligase
A
- enzyme that facilitates the joining of DNA strands together by catalyzing the formation of phosphodiester bonds
12
Q
vector: bacterial plasmids (5)
A
- 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
13
Q
vectors: viral vectors (3)
A
- 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
14
Q
what are the basic features a plasmid should contain (3)
A
- bacterial origin of replication (Ori)
- selectable marker (eg. antibiotic resistance)
- multiple cloning site (MCS)
15
Q
additional features of plasmid (in addition to basic features) (4)
- general
- examples (3)
A
- 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
16
Q
basic plasmid feature: bacterial origin of replication
A
- ability to replicate independently of the bacterial chromosome
17
Q
basic plasmid feature: selectable marker
A
- easier to select and isolate transformed cells
18
Q
basic plasmid feature: multiple cloning site
A
- contains unique restriction sites for ease of cutting and pasting DNA sequences into plasmids
19
Q
higher copy plasmids (2)
- definition
- advantage
A
- more copies of the plasmid per cell
- more material to work with and many more copies of desired DNA product per cell
20
Q
what are some applications of plasmids? (4)
A
- ease of selection for successfully cloned sequences
- promoter traps/enhancer traps
- manipulate genes in bacteria for expression in yeast
- create transgenic plants
21
Q
PCR components (4)
A
- DNA template with sequence of interest
- dNTPs
- primers
- polymerase
22
Q
PCR basic steps (4)
A
- denaturation
- annealing (of primers)
- extension (with dNTPs by polymerase)
- repeat cycles
23
Q
why do we need to”cut” and “paste” DNA pieces together? (4)
A
- for study of genes and genetic elements
- to engineer organisms for study/use
- mass production of useful biological molecules
- for gene therapy
24
Q
molecular cloning (2)
A
- involves a collection of methods that allow DNA to be inserted into vectors that can be grown in bacteria
- an alternative to PCR
25
what do we do with cloned DNA (2)
- can be manipulated for study
| - for production
26
advantages of molecular cloning
- does not require knowledge of the sequence to be cloned; each clone generates a different sequence
27
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
28
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
29
disadvantages of PCR
- need to KNOW THE SEQUENCE in order to design the PCR primers
30
insulin
- hormone secreted by the pancreas to regulate glucose uptake from the blood (blood sugar level)
31
type 1 diabetes
- failure to produce insulin (through mutation or destruction of pancreas cells that produce insulin)
- now controlled by insulin injections
32
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
33
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
34
steps in cloning human insulin gene
1. cut open vector with restriction endonuclease
2. ligate cDNA for human insulin into restriction site
3. introduce into cultured cells
4. screen for the clone with the sequence of interest and culture that clone
5. purify human insulin from cells (E. coli) or from solution (from yeast cells)
35
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
