Chapter 6

Question 1

what are the 4 steps of cell based cloning?

Answer 1

cell based cloning

1. formation of recombinant DNA
2. transformation
3. amplification
4. isolation of recombinant DNA clones

Question 2

cell based cloning: formation of recombinant DNA

Answer 2

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

Question 3

cell based cloning: transformation

> how?

Answer 3

transformation

> combine recombinant DNA and host cells into transformants

Question 4

cell based cloning: amplification

> how?

Answer 4

amplification

> repeated division of each transformed cell

> celldeath of cells not containing recombinant DNA which is antibiotic resistant

>>> results in identical cell clones

Question 5

what are plasmids?

what are they called in genetic engineering?

Answer 5

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”

Question 6

what are restriction endonucleases?

Answer 6

restriction endonucleases

> enzyme that makes cut at internal phosphodiester bonds

Question 7

what is the difference between endonuclease 2 and 1/3?

Answer 7

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

Question 8

where does type 2 endonuclease cut?

Answer 8

type 2 cuts within sequences that are palindromes

> ends of cutout sequence: sticky

> both sticky ends tend to base pair with complementary sequences

Question 9

what are two types of host cells available?

Answer 9

host cells

1. bacteria

> either with plasmids or bacteriophage

2. yeast

> especially used for extremely large DNA fragments

Question 10

why is transformation difficult?

what is the solution?

Answer 10

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)

Question 11

how do you know whether transformation succeeded?

Answer 11

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)

Question 12

4 characteristics of a good vector

Answer 12

good vector:

1. is origin of replication
2. DNA and vector are restricted with the same enzyme or with enzyme that create the exact same sticky overhang
3. small and easy to handle
4. has a marker to identify cells

Question 13

what is expression cloning?

> disadvantage?

Answer 13

expression cloning: vector contains expression signals, such as promotors and regulatory elements

> disadvantage: bacteria lack the enzymes needed for post translational processing

Question 14

what is an application of recombinant DNA techniques?

Answer 14

many products for treatment of disease or vaccination are produced using those techniques

Question 15

what is polymerase chain reaction used for?

Answer 15

cell free cloning

Question 16

what are the 3 steps of PCR?

Answer 16

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

Question 17

how can DNA be sorted on size using Gel electroforese?

Answer 17

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

Question 18

what are the advantages of PCR?

what is required?

Answer 18

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

Question 19

what is a ladder?

Answer 19

a mixture of DNA fragments of know size in a gel

Question 20

how can ladders be used in DNA fingerprinting?

Answer 20

compare known DNA fragment ladder to other DNA fragment ladders

> can be used in crime scene investigation

Question 21

what are 3 problems with DNA evidence?

Answer 21

DNA evidence

1. probability that different people have the same pattern?
2. DNA crime scene: dirty, degraded, mixture of more than 1 person
3. found DNA of person at crime scene -> does not mean they commited the crime

Question 22

what are 4 possible applications of PCR?

Answer 22

applications of PCR

1. medical: genetic testing for mutations

> discriminate between disease-causing mutation and normal allele

2. diagnosis of infectous diseases
3. forensic applications: genetic fingerprinting
4. research

> generating hybridization probes, DNA sequences, cloning and many more

Question 23

PCR vs cloning

> advantages/disadvantages

Answer 23

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

Question 24

what is RT-PCR?

Answer 24

RT-PCR: reverse transcripte polymerase chain reaction

> polymerase chain reaction using cDNA gained from reverse transcribing a RNA sample

Question 25

why does the melting temperature/primer annealing temperature depend on base composition of the DNA used?

Answer 25

GC base pairs have 3 hydrogen bonds,

AT base pairs only have 2

>> DNA with high GC base content is more difficult to separate