Chapter 6 Flashcards
what are the 4 steps of cell based cloning?
cell based cloning
- formation of recombinant DNA
- transformation
- amplification
- isolation of recombinant DNA clones
cell based cloning: formation of recombinant DNA
formation of recombinant DNA
> create homogenous vector DNA from bacterial cells containing extra chromosomal replicon (vector)
> create heterogenous target DNA sequences from the cells containing DNA that has to be cloned
> merge in to recombinant DNA
cell based cloning: transformation
> how?
transformation
> combine recombinant DNA and host cells into transformants
cell based cloning: amplification
> how?
amplification
> repeated division of each transformed cell
> celldeath of cells not containing recombinant DNA which is antibiotic resistant
>>> results in identical cell clones
what are plasmids?
what are they called in genetic engineering?
plasmid
> a small piece of DNA (often circular) that coexists with the main chromosome in a bacterial cell
> they carry genes that are not usually present in the main chromosome (e.g. antibiotic resistance)
> are able to transfer from one cell to another
> in genetic engineering: “vectors”
what are restriction endonucleases?
restriction endonucleases
> enzyme that makes cut at internal phosphodiester bonds
what is the difference between endonuclease 2 and 1/3?
endonuclease 2: binds at specific sequence of DNA molecule and makes a double stranded cut
endonuclease 1/3: no control over position of cut relative to specific sequence in the DNA molecule
where does type 2 endonuclease cut?
type 2 cuts within sequences that are palindromes
> ends of cutout sequence: sticky
> both sticky ends tend to base pair with complementary sequences
what are two types of host cells available?
host cells
- bacteria
> either with plasmids or bacteriophage
- yeast
> especially used for extremely large DNA fragments
why is transformation difficult?
what is the solution?
transformation
> the plasma membrane of cells is only selectively permeable, large molecules (DNA fragments) are normally unable to cross the membrane
> > > solution: electroporation
> cells incubated in calcium chloride solution and exposed to short high voltage pulses, which makes them “competent” (being able to take up foreign DNA from extracellular environment)
how do you know whether transformation succeeded?
by using a marker gene on your vector
> e.g. antibiotic resistance: bacteria that contain your vector are resistant, the rest not
> lacZ gene (blue/white color system)
4 characteristics of a good vector
good vector:
- is origin of replication
- DNA and vector are restricted with the same enzyme or with enzyme that create the exact same sticky overhang
- small and easy to handle
- has a marker to identify cells
what is expression cloning?
> disadvantage?
expression cloning: vector contains expression signals, such as promotors and regulatory elements
> disadvantage: bacteria lack the enzymes needed for post translational processing
what is an application of recombinant DNA techniques?
many products for treatment of disease or vaccination are produced using those techniques
what is polymerase chain reaction used for?
cell free cloning
what are the 3 steps of PCR?
PCR:
step1: denaturation - 1 minute/94C
> strand separate
step2: annealing - 45sec/54C
> forward and reverse primers bind to strands
step3: extension - 2 min/72C
> new DNA strand are synthesized
how can DNA be sorted on size using Gel electroforese?
sort DNA by size
> DNA is negatively loaded due to phosphate group
> migrates through the gel towards the positive pole
> small pieces migrate faster than larger pieces
what are the advantages of PCR?
what is required?
PCR
> straightforward method (quick)
> with smart enzyme mixtures fragments up to 10-20kb can be amplified easily
> just a small amount of DNA is necessary to start with
but
the sequence of boundary regions must be known
what is a ladder?
a mixture of DNA fragments of know size in a gel
how can ladders be used in DNA fingerprinting?
compare known DNA fragment ladder to other DNA fragment ladders
> can be used in crime scene investigation
what are 3 problems with DNA evidence?
DNA evidence
- probability that different people have the same pattern?
- DNA crime scene: dirty, degraded, mixture of more than 1 person
- found DNA of person at crime scene -> does not mean they commited the crime
what are 4 possible applications of PCR?
applications of PCR
- medical: genetic testing for mutations
> discriminate between disease-causing mutation and normal allele
- diagnosis of infectous diseases
- forensic applications: genetic fingerprinting
- research
> generating hybridization probes, DNA sequences, cloning and many more
PCR vs cloning
> advantages/disadvantages
PCR vs cloning
> PCR more efficient
> cloning costs more
> PCR is faster (4-5h vs 2-4 days)
> PCR is fully automated
but
> for PCR sequence information is needed to construct primers
> size of amplification product is smaller in PCR
what is RT-PCR?
RT-PCR: reverse transcripte polymerase chain reaction
> polymerase chain reaction using cDNA gained from reverse transcribing a RNA sample
why does the melting temperature/primer annealing temperature depend on base composition of the DNA used?
GC base pairs have 3 hydrogen bonds,
AT base pairs only have 2
>> DNA with high GC base content is more difficult to separate
