2960 Lecture 33

Recombinant DNA technology !

Molecular Cloning Genomic Libraries cDNA Libraries Sub-cloning

A clone !

A population of identical items that are derived from a single progenitor!

- we can use DNA molecules to do studies

Items could be:!

Genetically identical bacterial cells (a colony)! !Or" Genetically identical individuals ( the sheep Dolly)! molecular cloning technology allows production of a large quantity (many copies) of a particular DNA molecule!

Or DNA molecules!

Molecular cloning technology allows production of a large quantity of a particular DNA molecule.! How is it done? Why is it useful?

How is it done?!
1

2 steps:!

Create chimeric recombinant DNA molecules in a test tube by a Cut and Paste mechanism (vector + cargo DNA)! vector = DNA capable of autonomous replication in a host cell! cargo = any DNA from any source! Isolation and amplication of recombinant molecules in a host that supports growth of the vector!

How is it done?!

First focus on step 1!

Cut and Paste mechanism !

vector! DNA!

cargo! DNA!

recombinant DNA! vector-cargo! ! A Chimera !

What is a vector and why is it drawn as circular DNA?!

Requirements of a vector for molecular cloning:! Capable of autonomous DNA replication after introduction into a host cell! Capable of carrying foreign DNA ! = tolerates the insertion of extraneous DNA at one or more positions! Contains at least one genetic marker (selectable or screenable) to indicate vector s presence in the host! Genome that is well-characterized, of a limited size, & a single nucleic acid molecule!

- there are also vectors that can be used in animal cells - marker can be resistence to a drug, orescence, etc -

The most popular molecular cloning vectors are based on plasmids !

Double-stranded circular DNA molecules that replicate independently of the bacterial chromosome! Examples:! Resistance or R factors: plasmids! conferring antibiotic resistance! Fertility or F factor: ~100 kb plasmid in E. coli that is responsible for bacterial mating !

- there's only one F plasmid per cell -

An example of a plasmid cloning vector; one based on a multiple copy origin of replication!

Selectable marker (ampicillin resistance)!

Insertion sites for foreign DNA (<~10kb)!

Origin of replication! (multiple copy-from resistance plasmid)!

replicate independently of the bacterial chromosome Plasmid DNA ( ) is introduced into a cell by transformation direct delivery of DNA into a cell
Plasmid" GROWTH!

Plasmids: double-stranded circular DNAs that

- transformation: inserting plasmit into cell - as the cell grows you get not only more cells, but multiple plasmids

bacterial cell with circular genome

transformation!

after growth!

Amplification !

A BAC plasmid has an origin (from F factor) that replicates at one copy per cell-allows stable maintenance with large fragments of human DNA (>100kb).!

- not all plasmids produce multiple copies per cell - with these it is difcult to put large amounts of DNA in these plasmids - vectors with F factor origin only have one copy per cell - v important in tech used to sequence human genome

iGenetics Fig 8-6a"

Molecular Cloning!
Cut and Paste mechanism !

vector! DNA!

cargo! DNA!

recombinant DNA! vector-cargo!

How are the DNA molecules cut and pasted?!

DNA is cut using Restriction Endonucleases!

Restriction Endonucleases (or Restriction Enzymes) are proteins produced in micro-organisms that recognize specic DNA sequences and cleave both sugar-phosphate backbones at specic positions relative to their recognition sequence.! Many (but not all) restriction endonucleases recognize:! * 4-6 bp sequences! * palindromic sequences! "(same sequence read 5 !3 on both strands)!

- often sequences are BOTH 4-6 bps and palindromic (but not always) EcoRI binds as a dimer w/ palindromic recognition sequence - all restriction enzymes cleave 5' phosphates and 3' hydroxyl gps

Example: EcoRI!

5 -pAATTC! 5 -GAATTC-3 ! GOH! +! HOG! 3 -CTTAAG-5 ! CTTAAGp-5 !

Restriction enzymes protect cells from infection by viruses (bacteriophages) and from introduction of foreign DNA. Host DNA is protected from cleavage by DNA methylation of the cutting site.!

- restriction sites within its own DNA has been protected by methylation

A large number of different restriction endo-nucleases are available from commercial vendors!
www.neb.com!
Website for New England Biolabs, a major commercial source of restriction enzymes!

N = A,G,C, or T! W = A or T!

The Multiple Cloning Site (MCS) has recognition sites for many restriction enzymes. The cut the plasmid once and each of them is 6 or 8bp in extent.!

- each restriction enzyme cuts plasmid once

Calculating the restriction enzyme cleavage frequency!

Restriction endonucleases recognize short sequence motifs; usually 4-6 bp sequences! What is the expected frequency of a particular restriction enzyme cleavage site? Assume the probability of a specic base at any position to be random = 1/4!

5 -G A A T T C-3 ! 3 -C T T A A G-5 !
1 1 1 1 1 1 x x x x x = 1 4 4 4 4 4 4 4096

Example: EcoRI!

Molecular Cloning!
EcoR1! + EcoR1 Enzyme!

?!
R! R!

?!

First step in molecular cloning: A plasmid vector with a single EcoR1 cutting sites is opened up after incubation with EcoR1 enzyme.!

Restriction Endonucleases(cont.)!
Example: EcoRI!
6 bp palindromic! recognition sequence!

Generates a 5 overhang of 4 nucleotides!

GAATTC CTTAAG
3 OH

G CTTAAp

5 Phosphate

pAATTC

5 Phosphate

3 OH

10

All DNA ends generated by EcoRI digestion have the same structure, regardless of the source of the DNA! All EcoRI ends are compatible & sticky the overhangs are complementary and they can be joined together!
Vector"

GOH pAATTC CTTAAp HOG

Cargo"
p

AATTC HOG

...

GOH CTTAAp

DNA ligation
vector end!
3 OH

G CTTAAp

5 Phosphate

cargo end!

pAATTC

5 Phosphate

3 OH

DNA ligase + ATP

- incubated together with DNA ligase - generally ligase obtained from bacteriophage T4 - all restriction enzymes leave 5' phosphates, DNA ligase reqs

GAATTC CTTAAG
recombinant!

DNA ligase:! * Reform sugar-phosphate backbone on both strands! * Requires 5 -Phosphate & 3 -OH!

11

DNA Ligase:

creates a phosophodiester bond between adjacent 5 -phosphate and 3 -OH

DNA ligase

3 -OH

5 - P

Berg et al. Biochemistry (6th edition) Fig. 28-22

EcoRI

EcoRI

EcoRI

- distribution of fragment sizes

EcoRI

drugR plasmid vector

EcoRI

EcoRI

target DNA (e.g. yeast or human)

EcoRI

drugR

12

drugR!

mix and ligate a b

- most probably problematic outcome is that the DNA reseals - you can force recombinance by a high concentration of cargo, but then the cargo fragments can ligate to each other

Can other outcomes occur???!

Yes but techniques have been developed to keep these alternative outcomes to a minimum!

Preventing the digested vector DNA from recircularizing!

EcoRI!
3 OH

- use phosphatase which eliminates 5' phosphate (req for DNA ligase)

drugR! plasmid vector!

EcoRI

G CTTAAp

5 Phosphate

pAATTC
3 OH

5 Phosphate

Phosphatase!
3 OH

G CTTAA
drugR!

5 OH

5 OH

AATTC G

3 OH

These ends cannot be ligated to each other because 5 PO4 removed!

13

Can a phosphatased end be used for DNA ligation?! Yes, if ligated to an end with a 5 -PO4 !
3 OH

G CTTAA
Phosphatased! vector DNA!

5 -PO4

pAATTC
3 OH

5 OH

G
target DNA!

DNA ligase + ATP!

G AATTC CTTAA G
Ligation on one strand but not the other; molecules are covalently joined!

- if it gets ligated to an end that has a 5' phosphate on one end, it can still be ligated, is ligated on one end, and then later the bacteria seals the knick - the longer, more precise the recognition site, generally the larger average fragment site - a blunt end doesn't have sticky ends, can be ligated to any blunt end

drugR!

Only the first step

c

mix and ligate a

Still in a test tube!

b

drugR! drugR! drugR!

14

Molecular Cloning takes 2 steps

1

Create recombinant DNA molecules in a test tube by a Cut and Paste mechanism (vector + cargo DNA)
vector = DNA capable of autonomous replication in a host cell cargo = any DNA from any source

Isolation and amplification of recombinant molecules in a host that supports growth of the vector

The second step allows production of large amounts of a particular recombinant DNA molecule in isolation
Mixture of recombinant vector-cargo chimeric DNA molecules in a test tube transformation into a host cell/organism Identification & recovery of independent transformation events Individual clones have a different recombinant molecule in an amplified form

- host cell is generally E. coli

cloning

15

a c drugR drugR Mixture in a test tube c insert a insert b drugR

transformation into a host cell/organism (e.g., E. coli)

- can treat E. coli with calcium, electric shock to allow molecules to get into cells - plasmid has drug resistance - cells can have different numbers of plasmids

Each bacterial colony represents an independent clone

Petri dish with nutrient media + drug Untransformed host cells die; a selection!

From this point, you can grow an unlimited amount of a particular clone = population of identical cells carrying the same recombinant DNA molecule ! Purify DNA Now in a position to analyze & manipulate the structure of a particular DNA fragment that has been cloned

- can study gene itself, binding site, sequence that codes for a protein, etc

16

Step back from the specifics

- DNA always behaves the same

What makes molecular cloning possible?!

Conserved DNA structure Restriction endonuclease that act like molecular scissors and generate compatible ends Ability to join (ligate) independent DNA molecules Identification and detailed characterization of vector molecules Ability to introduce vector molecules into host cells

Genomic Library concept

Break a large genome into smaller pieces in the form of independent recombinant DNA clones genomic clones = analogous to books

- librarys are collections of fragment clones -

A collection of independent clones constitutes a library, which collectively represents the original larger genome

17

Consider the human genome: 23 chromosome pairs

Human karyotype G-banding Human genome 3x109 bp/n (3 million kb)!

http://www.selu.com/bio/cyto/human/index.html!

Each chromosome contains an extremely long piece of DNA

Most cloning vectors can hold only a very small cargo (<100 kb)

chromosome

Decondensed dsDNA

18

Agarose gel electrophoresis of EcoRI digested human DNA! Size markers Human DNA / EcoRI

12 kb

2 kb 1 kb

The EcoRI digested human DNA looks like a continuous smear because there are so many different restriction fragments in the sample!

migration

DNA is visualized by uorescence after staining with the intercalating agent, ethidium bromide!

Agarose gel electrophoresis of EcoRI digested DNA of 5 independent clones from a genomic library! Vector! DNA! Insert DNA !

Independent Size! Size! marker genomic clones! marker s! s! A! B! C! D! E!

Grifths; Fig. 11-13!

19

Genomic libraries are useful if you want to retrieve or decipher all the genetic information in an organism, but in eukaryotes this information is fragmentary and dispersed.! If one is interested in genes = coding sequences for genetic engineering, then how do you put this fragmentary information back together efciently?!

- euks have introns/ exons and genomic clones are sometimes not useful

Transcription of a mammalian PolII gene!

1! ! ! primary transcript! ! 2! ! 2! 3!3! 3!3!

- take advantage of the fact that mRNA's are processed and make copies of mRNA

[! ppp!

1!

]!
AAAAAA!

Capping! 7meGppp! 1! 2! ! Splicing! ! 2! ORF!

Cleavage/PolyA Addition! 3!3!

processed transcript 7meGppp! 1! or mRNA!

5 UTR!

3!3!
3 UTR!

AAAAAA!

20

cDNA = complementary DNA or copy DNA! (DNA version of RNA)!

processed transcript!
ATG

ORF
AAAAAAA

- have to purify mRNA from all the RNA: use polyA tail (attach to oligo dT) and extract mRNA - make of mRNA using reverse transcriptase isolated from retrovirus

First strand cDNA synthesis!

1. Afnity purify poly(A)+ RNA (i.e., mRNA) on oligo d(T) column/beads! 2. First strand cDNA synthesis using reverse transcriptase (RT), dNTPs and an oligo d(T) or a gene-specic primer!

Synthesis of cDNA (for library)!

HOGp HOGp

mRNA

AAAAAAAA AAAAAAAA Anneal dT primer TTTTTTTTT AAAAAAAA Extend cDNA to 5' TTTTTTTTT end of mRNA AAAAAAAA Add 3' tail with TdT TTTTTTTTT

- this is just one way to make cDNA - TdT generates polyG tail without template - use polyC tail to attach to Gs and then generate the other strand using DNA polymerase

HOGp

HOGp

TTTTTTTTT

Eliminate RNA

CCCCCCCCC

cDNA

AAAAAAAA TTTTTTTTT

Anneal 5' primer and extend with DNA polymerase

21

Double-stranded cDNA can be ligated into vectors to generate cDNA clones!

CCCCCCCCC

cDNA

AAAAAAAA TTTTTTTTT

! cDNA library!
The representation of a given gene in a cDNA library is determined by mRNA abundance in the tissue/cells from which the RNA was puried!

- different genes expressed at different levels - not commonly expressed genes may only have a few copies of cDNA - non-expressed genes will have no cDNA - presence of cDNA is directly correlated to the abundance in nature - can use cDNA to make proteins from mammalian genes, etc

Genomic DNA library!

Inserts = segments of DNA as it exists in the native genome!

cDNA library!
Inserts = DNA copy of processed RNA transcripts!

22

Molecular Sub-Cloning! Transfer a sub fragment/sequence from a clone of a larger fragment into a vector for the purpose of studying it. Example: from a clone of 25kb fragment of yeast DNA containing the yeast gene for Tata Binding Protein (TBP), we want to sub-clone a smaller fragment containing only the TBP gene. How do we do it?!

2000 3> 2> 1> <1 <2 <3 2000

4000

6000

8000

10K

12K

14K

TBP ORF"
16K

18K

20K

22K

24K 3> 2> 1> <1 <2 <3

4000

6000

8000

10K

12K

14K

16K

18K

20K

22K

24K

- TBP is a small part of the insert - look for restric enzyme cutting sites that ank the gene

Spe I 26302

4882 Bam HI 5065 Bam HI

Vector+ChrVDNA.xdna

Vector+ChrVDNA.xdna

27642 bp
Bam HI 19337

27642 bp

TBP"

TBP"

Bam HI 18670 9593 Spe I Spe I 17425

A 25 kb fragment of yeast DNA cloned into a vector contains the yeast TBP gene. How do we get it out as a smaller fragment?"

Break it up into sub-clones using additional restriction enzymes, in this case BamH1 ( G^GATCC! ) and " CCTAG^G! Spe1( A^TCAGT!), with six base TAGTC^A! recognition sites and non-compatible 4 bp 5 overhangs"

23

The Multiple Cloning Site (MCS) has recognition sites for many restriction enzymes. Each of them is 6 or 8bp in extent.!

Use vector sites compatible with cargo anking sites!

Molecular Sub-Cloning!
BamH1! Spe1!

B! B! S!

S!

- we have to isolate the fragment we want - don't have to phosphatase because B and S ends are not compatable

S" B" B" B"

B" S"

S"

First step in molecular sub-cloning: A sub-cloning plasmid vector with single BamH1 and Spe1 cutting sites is opened up after inucubation with the enzyme(s) and a clone is cut up into BamH1/Spe1 fragments.!

24

Molecular Sub-Cloning (cont.)!

S!

S!

S!

B! B!

B! Join fragments with DNA ligase and ATP. Only compatible ends are joined together.!

Isolate vector and TBP gene containing fragments on agarose gel!

Cut fragments out of gel and mix together!

Molecular Cloning technology allows the isolation, amplication, and manipulation of specic DNA fragments from large, complex genomes! ! AND so ! does PCR!

- PCR and molecular cloning do similar things

Grifths; Fig. 11-1!

25

Molecular Cloning technology allows the isolation and amplication of specic DNA fragments from a large, complex genomes and so does PCR. What are the pros & cons of each technique?! PCR! Molecular Cloning!
Ease! Speed! Error-prone! Prior seq knowledge required! Size constraints (<10 kb)! Flexible-manipulation!! Generally high-delity! No prior seq knowledge needed! Size constraints less! Time-consuming; multi-step!

- error of PCR depends on polymerase you use

26