Lecture 30 - DNA cloning and experimental gene expression

Question 1

Goal of DNA cloning

Answer 1

produce large quantities of identical DNA

Question 2

General scheme of DNA cloning

Answer 2

Vector + DNA fragment (called insert) give recombinant DNA (any DNA from diff. sources) -> replicated within host cells and manipulated

Question 3

What is a plasmid + length without essential sequences

Answer 3

Small circles of DNA in prokaryotes, indepedent of big circular chromosomes. w/ essential seq : 1.2 - 3kb

Question 4

3 important regions on plasmid

Answer 4

Origin or replication, Selectable marker, Polylinker

Question 5

What is a polylinker

Answer 5

Region with multiple restriction sites (or ‘‘multiple cloning site’’) where insert added

Question 6

What is a selectable marker

Answer 6

An antibiotic resistance gene (like for ampicillin or kanamycin)

Question 7

Steps of DNA fragment insertion (3)

Answer 7

Cut vector with restriction fragment, insert the DNA fragment, DNA ligase seals DNA:DNA joints

Question 8

What is common to all restriction sites and definition of that

Answer 8

palindromic sequence. Same sequence from 3’->5’ on both strands

Question 9

Most known restriction enzyme and restriction site sequence

Answer 9

EcoRI. Restriction site sequence is (from 5’ to 3’) GAATTC

Question 10

2 ways of cutting a restriction site + example for each

Answer 10

Leave sticky ends (EcoRI) or blunt cut (SmaI)

Question 11

Usual sequence length for a restriction site and frequency of 6 bp sites in the genome

Answer 11

4-6 bps, restriction site not hard to find in genome -> one 6 bp sequence every 2-3 kb

Question 12

Condition for proper insertion of insert in restriction site and ex. w/ EcoRI

Answer 12

Must have complementary sticky end. If EcoRI has cut, need AATT (the sticky end sequence from 5’ to 3’)

Question 13

Which DNA ligase is common for ligation part and how it uses energy

Answer 13

T4 DNA ligase. Will convert 2 ATP molecules to 2 AMP +PPi

Question 14

What transformation or transfection means

Answer 14

Putting recombinant DNA in the bacteria

Question 15

What is cloning ultimately

Answer 15

Being successful in putting one recombinant DNA molecule in a cell

Question 16

why cloning is difficult

Answer 16

Difficult cause cells don’t like taking up plasmid

Question 17

Secret to cloning (solution to the difficulty)

Answer 17

Use very dilute recombinant DNA (small number of molecules) so that just a few cells take up one and it is impossible for a cell to get 2

Question 18

Where bacteria are cultured and characteristic of milieu + why

Answer 18

nutrient agar plates. contain ampicillin to kill bacteria that didn’t take up plasmid

Question 19

After many colonies of bacteria containing the plasmid are obtained, how is plasmid purified

Answer 19

One clone is picked, put in liquid culture and then plasmid DNA is purified from bacteria

Question 20

Goal of a DNA library + something particular about genes in a DNA library

Answer 20

Produce many copies of all genes. Genes in DNA library only have their exons

Question 21

Technique similar to cDNA libraries (but that doesn’t allow multiplication of many genes)

Answer 21

RT-PCR

Question 22

Step 1 of making DNA library

Answer 22

Get an mRNA mixture from cell extract and produce a cDNA mixture using reverse transcriptase

Question 23

Step 2 of making DNA library (after we have cDNA)

Answer 23

Make dsDNA out of each cDNA and each dsDNA is an insert that will be put in recombinant DNA (plasmid)

Question 24

Step 3 of making DNA library (after plasmids done)

Answer 24

Transform bacteria with plasmids containing our cDNA and grow colonies

Question 25

Step 4 of making DNA library (after we grew colonies)

Answer 25

Each colony contains cells with one of our cDNA, pick colony and isolate plasmid

Question 26

2 broad reasons for expressing foreign proteins in bacteria or in eukaryotic cells

Answer 26

Biotechnology and Research

Question 27

Biotechnology : Why express foreign proteins in bacteria or in eukaryotic cells

Answer 27

Produce large amounts of a protein that is rare in nature or difficult to purify or expensive to purify or ethical issues for human sources

Question 28

Research : Why express foreign proteins in bacteria or in eukaryotic cells

Answer 28

Express wild-type,mutant or fragment of a protein to study function in a cell

Question 29

What is necessary to drive transcription of the cDNA insert now in a plasmid

Answer 29

Need a promoter

Question 30

Restrictions about promoters used for expressing cDNA in a plasmid

Answer 30

Must use prokaryotic promoter if in a prokaryote

Eukaryotic promotor of the specific cell type if in a eukaryote

Question 31

Common prokaryotic promoter + it is required for transcription of WHAT

Answer 31

LacZ promoter. For transcription of an operon of 3 genes, including LacZ gene

Question 32

What lacZ gene (part of the operon) codes for + function and how LacZ promoter is regulated

Answer 32

LacZ gene codes for beta-galactosidase, prot for lactose metabolism. LacZ promoter regulated by lactose qt.

Question 33

In transfected bacteria, what substance is used to regulate lacZ promoter expression

Answer 33

IPTG, a lactose analogue

Question 34

When inserting our cDNA in a plasmid containing LacZ promoter, what do we have to do (we want to express OUR protein)

Answer 34

Remove LacZ gene

Question 35

2 types of transfection in eukaryotic cells/organisms

Answer 35

1) Transient transfection

2) Stable transfection

Question 36

What happens to plasmid in transient transfection

Answer 36

Goes in nucleus, many cells take it up but it can’t replicate fast enough

Question 37

Transient transfection : What happens w/ time to level of protein (of our cDNA) expression

Answer 37

Less cells have the plasmid so less and less protein expression

Question 38

How eukaryotic cells in culture are transfected (2 methods)

Answer 38

Lipid treatment

Electroporation

Question 39

In eukaryotic transfection, what selectable marker is used and what drug is put on the plate to kill cells without plasmid (STABLE TRANSFECTION)

Answer 39

Neomycin resistance gene (cause neomycin kills eukaryotes). G-418 put on plate (a neomycin analogue)

Question 40

Obtaining transgenic mice : Step 1

Answer 40

Inject foreign DNA in one of the pronuclei (2 nuclei of zygote - 2 fused gametes) and transfer injected eggS into foster mother

Question 41

How DNA injected inserts in pronuclei DNA

Answer 41

High-change of random insertion by non homologous end joining

Question 42

Effect of random insertion of foreign DNA in pronuclei DNA on genes already there

Answer 42

Most random insertions don’t disturb other genes, but SOME MAY

Question 43

Obtaining transgenic mice : Step 2 (what happens after injected eggs are in foster mother)

Answer 43

10-30% of offspring contain foreign DNA in chromosomes of all their tissues AND GERM LINE

Question 44

Obtaining transgenic mice : Step 3 (we have offspring with the foreign DNA in chromosomes of tissues and germ line, what next)

Answer 44

Breed mice expressing foreign DNA to propagate DNA in germ line

Question 45

Difference between transient and stable transfection

Answer 45

Stable transfection uses selectable marker so only cells that have and express our cDNA survive