Molecular Bio Unit 3

Front 1

Cloning

Back 1

isolation of specific DNA fragment of interest and propagating identical copies (clones) into a suitable vector

Front 2

Gel electrophoresis

Back 2

2. Used to separate DNA, RNA or proteins according to mass
3. Fractionation based on molecular weight
4. Cannot compare different forms

Front 3

Most common gel for electrophoresis

Back 3

agarose and polyacrylamide

Front 4

Cloning

Back 4

constructing a recombinant DNA molecule and maintaining it in a cell

Front 5

Vector

Back 5

must have Ori site
Dna molecule containing information needed to propagate the cloned DNA in host cell

Front 6

Insert

Back 6

the piece of DNA put into vector

Front 7

Steps for Cloning

Back 7

1. Restriction enzyme
2.Phosphotase (prevent religation)
3 Provide DNA ligase.
4. transformation into E. coli
5. select with antibody
6.screen for gene (extract, digest)
7. electrophoresis
8. sequence to confirm

Front 8

why are restriction enzymes so useful?

Back 8

1. They cleave at a specific sequence
2. The overhang/sticky end can be 5’ or 3’
3. a blunt end

Front 9

What must a restriction enzyme leave?

Back 9

3' OH for DNA ligase

Front 10

3 Ways to ligate inserts into vectors

Back 10

1. Overlapping/ cohes, sticky ends
2. Blunt ends
3. Ligating incompatible ends

Front 11

How do you ligate incompatible ends?

Back 11

a. Treat with klenow DNA pol I (modified Pol I lacking 5’ -3’ exonuclease)

Front 12

Bacterial Transformation

Back 12

ChemicalL CA2+
Electrophoration: DNA + open pores on the electric field

Front 13

Animal cells transformation

Back 13

a. Ca DNA treatment of cells
b. microinjection of embryos/ eggs
c. electrophoration

Front 14

Fungi transformation

Back 14

1. remove cell wall: Ca2+
2. electroporation

Front 15

Plant transformation

Back 15

a. Microprojectile bombardment
b. Agrobacterium mediated

Front 16

Types of vectors

Back 16

Plasmids (small circular bacterial replicons with Ori)
Viruses (bacteriopage: linear or circular DNA)
Linear artifical chromosomes (yeast)

Front 17

4 Desirable characteristics of vector

Back 17

1. replicates stably
2. easily introduced
3. polylinker ( restriction enzyme sites to insert cloned DNA)
4. selection marker

Front 18

what is the most common way to select btwn gene of interest and gene without

Back 18

Electrophoresis

Front 19

How do you prevent inset minus vectors?

Back 19

1. use two different enzymes with different sticky ends to reduce ligase efficiency
2. phosophatase: dephosphorlyate

Front 20

Expressed vector

Back 20

has gene of your interest

Front 21

“blue-white screen” using β-galactosidase (lacZ gene)

Back 21

White: LacZ = has been disrupted
Blue: no insert

Front 22

Restriction Mapping

Back 22

1. restriction enzyme
2. electrophoresis
to confirm close structure

Front 23

Types of cloning

Back 23

1. Isolation of a desired piece of DNA from the whole genome
2. Make modifications to piece of DNA
a. Placing a human gene in front of promoter to make protein in bacteria
3. Make a research tool

Front 24

Library

Back 24

a collection of genomic fragments, which encompasses the entire genome
a. The genome is split up into small cloned DNA to search for individual genes
b. The library is propagated (make many copies) in vectors (plasmids or bacteriophage) so that there can be many copies of the library
c. Individual bacteria(pages) are identified and purified for studies

Front 25

Strategies for cloning a gene from a genomic library

Back 25

1. Use a DNA prode
2. Based on protein product
3. Based on function
4 Based on genetic location

Front 26

Cloning a gene from yeast

Back 26

1. Extract DNA
2. Fragment DNA
3. Ligate into vector
4. Transform the host
5. “library”
6. Identify desired clone

Front 27

Cloning actin from penicillium yeast

Back 27

1. Extract DNA from penicillium spores
2. Fragment DNA
3. Ligated into vector
4. Transform E. coli
5. Library
6. Colony Life and hybridize

Front 28

Colony Life and hybridize

Back 28

a. Tranfer to nylon or nitrocellulose membrane
b. Lyse the bacteria with NaOH
c. DNA replicated in colonies on membrane
d. Soak with 32P radioactive actin probe (heterologous probe from yeast)
e. Wash off the unbound probe
f. Expose to film

Front 29

Making radio-label probes for southern blotting

Back 29

1. In vitro transcription (makes labeled RNA with RNA Pol)
2. End labeling (add radioactive phosphate to 5’ ends of DNA using polynucleotide kinase)
3. Nick-translationg (gap repair using DNA pol ! and radioactive dNTPs)
4. Random Hexamer-primer synthesis (DNA pol 1)

Front 30

Cloning E coli DNA Pol α Subunit

Back 30

1. purify protein from gel, column fraction
a. produce antibodies:
i. screen expression library
b. sequence protein:
i. designing DNA Probe and screen DNA library
ii. use DNA sequence to figure out protein sequence

Front 31

Screening clones with antibody

Back 31

1. filter
2. blot proteins from plaques
3. filter with blotted protein
4. incubate with specific antibody, then with labeled protein A (staphylococcus protein that binds antibodies)
5. autoradiograph

Front 32

Cloning vectors based on Bacteriophage λ

Back 32

1. λ are better than plasmids for expressing proteins for library screening, because cells are lysed in the virus life cycle
2. λ is very easy to transform into E coli
3. λ libraries also larger inserts than plasmids

Front 33

4. plaques

Back 33

a localize abnormal path on a body part or surface
a. a clear area in a culture produced by destruction of cells by virus

Front 34

complemenation:

Back 34

make up for defect

Front 35

shotgun cloning

Back 35

massive transformation

Front 36

how do you perform restriction analysis of gene in uncloned genomic DNA?

Back 36

restriction digest and southern hybridization

Front 37

what are two ways to study genes

Back 37

1. remove gene (knock out)
2. overexpress gene

Front 38

DNA sequencing

Back 38

1. chemical cleavage
2. Sanger (dideoxy)

Front 39

Sanger

Back 39

dideoxynucleotide triphosephate" ends replication because there is no OH

Front 40

PCR requirements

Back 40

template DNA
oligonucleotide primers that flank target
DNA polymerase

Front 41

PCR 3 main steps

Back 41

Denaturing
annealing: Hybridize primer
elongation: DNA synthesis: Taq Pol

Front 42

Primer assistance

Back 42

3' OH must match
5' can have mismatch

Front 43

Application of PCR

Back 43

1. rapid lab tool
2. dna finger printing
3. identification of pathogens