Recombinant DNA Technology Dr. Hui LI Office : S408 Tel: 26538722.

Recombinant DNA

Technology

Dr. Hui LIOffice : S408Tel: 26538722

Topic 2

Ligation of target DNA and vector

DNA ligase (from E.coli or from T4 bacteriophage) is used to join the 5’ end (phosphate groupe) and the 3’end (hydroxyl group) of two fragments of DNA

1. Cohesive end ligation

1.1 ligation between two cohesive ends of DNA

1.2 ligation between a target DNA and a vector

With 1 enzyme of digestion=

2 possibilities of insertion

To prevent the self-annealing of the digested vector, alkaline phosphatase could be used.

ligase

nick

Directional cloning. DNA molecules whose ends have different overhangs can be used to form chimeric constructs in which the foreign DNA can enter the plasmid in only one orientation. The foreign DNA is digested with two different restriction enzymes (HindIII and BamHI ), and the plasmid is digested with the same two enzymes.

With 2 enzymes of digestion=

Only 1 possibility of insertion

5’5’

5’5’3’

3’

3’

3’-OH -OH

-P -P

P- P-

HO- HO-

5’

3’-OH-P

P-

HO-

5’

3’

2. Blunt end ligation

Blunt-end ligation is an alternative method for joining different DNAs . This method depends on the ability of phage T4 DNA ligase to covalently join the ends of any two DNA molecules. Attention: the reaction is much less efficient by rapport to that with sticky ends!

1972, P. Labban and P. Kaiser (Stanford University)

2.1 homopolymer tail-joining

Using terminal transferase the synthesis of homopolymer tails of the defined length at both 3' termini of double stranded DNA and vector is possible.

In the presence of precursor dATP, terminal transferase helps to add poly-dA at 3' termini of vector. Likewise the same enzyme adds poly-T at 3' termini of DNA molecule, when precursor TTP is present.

The linearized vector having tails is incapable of recircularization, unless ligated to a double stranded DNA fragment. The vector and DNA tails are annealed. The poly dA-dT tails are then ligated by T4 DNA ligase.

If poly dG-dC tails are used, instead of poly dA-dT tails, a high initiation temperature (up to 37C) is required for the annealing reaction. The major advantage in using poly dG-dC tails is that the hybrids are more stable than poly dA-dT hybrids.

Inconvenience

Advantage

Can join any given DNA fragments

difficult to retrieve the inserted DNA fragment due to the loss of recognition sites

非酶切位点

GGAATTCCCCTTAAGG

EcoRI linker：

2.2 Linkers

Linkers are the chemically synthesized double stranded DNA oligonucleotides containing on it one or more restriction sites for cleavage by restriction enzymes, e.g. Eco RI, Hind III, Bam HI, etc. Linkers are ligated to blunt end DNA by using T4 DNA ligase. Both the vector and DNA are treated with restriction enzyme to develop sticky ends. The staggered cuts i.e. sticky ends are then ligated with T4 DNA ligase with very high efficiency to the termini of the vector and recombinant plasmid DNA molecules are produced.

1 ） very easy to retrieve the inserted DNA fragment;

sometimes the inserted DNA contains the same restriction site

2 ） can make a Polylinker on a vector.

Inconvenience

Advantage

5‘p-GATCCCGG-OH3’ GGCC-p5’BamHI adapter

Adapters are also the chemically synthesized double stranded DNA oligonucleotides, with a blunt end at one side, and a cohesive end at the other side which contains a recognition site for a restriction enzyme.

Using T4 DNA ligase, adapters can be added to a blunt-ended DNA fragment

2.2 Adapter

Blunt-ended DNA

5’p-GATCCCGG-OH3’ HO-GGCC-p5’

BamHI adapter

P-

-P

5‘p-GATCCCGG- GGCC-

-CCGG-GGCCCTAG-P5’

5‘p-GATCCCGG- GGCC-

CCGGGGCCCTAG-P5’

CCGGGATCCCGG-OHGGCCCTAGGGCC-P5’

Self-ligation of the adapters

Self-ligation of the adapters

Countermeasure:

Treat the adapters with CIP(小牛肠碱性磷酸酶 ) to hydrolyse 5’-phosphate group. Before ligation with the vector, use T4 polynucleotide kinase to recover the phosphate.

Inconvenience

Advantage

No need to be digested before ligation

5‘p-GATCCCGG-OH HO-GGCC-P

P-CCGG-OHHO-GGCCCTAG-P5’

CCGGGATCCCGG-OHHO-GGCCCTAGGGCC5’

5‘GATCCCGG-OH HO-GGCC5’

5’CCGG-OHHO-GGCCCTAG5’

CIP

-OH

HO-

Blunt-ended DNA

5’p-GATCCCGG-OH3’ HO-GGCC-p5’

BamHI adapter

P-

-P

5‘GATCCCGG- HO-GGCC

CCGG-OH-GGCCCTAG5’

5’GATCCCGG-OH3’ HO-GGCC5’

nick

nick

HO-

-OH

CIP

T4 ligase

Even if the presence of the nicks, the adapters could still ligate with the DNA fragment

To pair with the multiple cloning sites of the vector;

To avoid the same restriction sites inside the DNA fragment

Structure:

3’: 15~20bp complementary with the template;5’: 6~10bp a restriction site of an endonuclease enzymeAND 3~5 bp more to give the restriction enzyme room to sit on the DNA

3. Ligation of PCR product3.1 To creat a restriction site at the end of 5’ of primes

PRIMER 1GCAGAATTCCOMPLEMENT

TEMPLATE

EcoRI site

5’- -3’

GCAGAATTCCOMPLEMENT5’- -3’

3’

TEMPLATE

PCR PRODUCT5’- -3’

3’

ANNEALING

EXTENSION

TEMPLATE ‘

TEMPLATE

3’

3’

GCAGAATTC

GCTAGCCGGCOMPLEMENT

TEMPLATE

BamH I SITE

-5’3-’

5’

ANNEALING

EXTENSION

TEMPLATE’

TEMPLATE

3’

3’

COMPLEMENT -5’3’-

TEMPLATE5’

PCR PRODUCT -5’3’-

PRIMER 2

GCTAGCCGG

GCTAGCCGG

GCAGAATTC5’--3’

GCTAGCCGG -5’3’- CGTCTTAAG

CGATCGGCC

EcoRI

BamHI

AATTC5’--3’

GCTAG -5’3’- G

C

EcoRI BamHI

（ 1 ） Normally, PCR product has an additional “A” at both sides of 3’

Characteristic feature of Taq DNA polymerase when running a PCR

（ 2) T-vector has a supplementary “T” at both sides of 3’  

3.2 To ligate directly with a T-vector

A

A

dNTP

T

T

dTTPTaq DNA polymerase

vectorPCR product

TA TA

1. 插入片断与载体的酶切位点互补

相同的粘性末端才能有效地连接。

尽量避免平端连接。

决不能进行非粘性末端连接。

What you need to know more...

EcoRI EcoRI EcoRI

（ 1）用相同的酶切

（ 2）用同尾酶切

BamHI 、 BclI 、 BglII 、 Sau3AI 、 XhoII

都产生 GATC4个碱基的粘性末端。

2. DNA插入的方向正确（ 1）用双酶切

由于产生的粘性末端不同，只能以固定方向连接。

EcoRI BamHI

EcoRI BamHI

EcoRIBamHI

3. 插入基因的开放阅读框（ ORF）正确（ 1 ） DNA定向插入

（ 2）起始密码

尤其当载体上有 ATG起始密码的时候，更要注意。

ATGGAATTC

载体

ATGCGGAATTCT

插入片断

EcoRI EcoRI

ATGGAATTC T

重组 但移码突变！

4. 防止载体自身环化连接（ 1）提高插入片断的用量

连接反应体系中，插入片断比载体多 10倍以上。

（ 2）用碱性磷酸酶处理载体

载体切口的 5’磷酸被除去，不能自我连接。但可以与插入片断以单链连接。

5’ 3’

载体

插入片断

1. 载体自身环化连接（能存活）

2. 载体之间互相连接（能存活）

3. 插入片段互相连接（不能存活）

4. 一个载体与几个插入片段重组（能存活）

5. 几个载体与一个插入片段重组（能存活）

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