Recombinant DNA2 kỹ thuật tái tổ hợp DNA bản dịch đính kèm

Phép lai “Southern” được đặt theo tên của Sir Edwin SouthernPhát triển vào năm 1975Một trong những ấn phẩm khoa học được trích dẫn nhiều nhấtGiúp Sir Southern đạt giải Lasker năm 2005 Đặt tên các phương pháp còn lại như là một kiểu chơi chữ theo Southern BlotSouthern blot : DNADNASử dụng gel điện di cùng với đầu dò lai đặc trưng cho mỗi đoạn cắt giới hạn của DNA genomic (hoặc DNA từ nguồn khác, chẳng hạn như plasmid)DNA xác định với trình tự base đặc trưngCó thể thực hiện để phát hiện những gene cụ thể tồn tại trong tế bào.Mục đích của phép lai SouthernCố định DNA vào một chất cố địnhMàng + chất nền giống giấy+ nylon hoặc nitrocellulose+ thường tích điện dương yếuNhận biết trình tự DNA (gene) quan tâmQuy trình chung của kỹ thuật Southern Blot

Heat shock Transformation

Electrophoration Transformation

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Southern Blot: DNA-DNA*

Uses gel electrophoresis together with hybridization probes to characterize restriction fragments of genomic DNA (or DNA from other sources, such as plasmids)

Identifies DNA with a specific base sequence

Can be done to detect specific genes present in cells

Goals of Southern Hybridization

• Immobilize DNA onto a permanent

substrate

• Membrane

– paper-like matrix

– nylon or nitrocellulose

– usually has a slight positive charge

• Identify DNA sequence (gene) of interest

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General Scheme for Southern Blot

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Southern Steps

1 DNA to be analyzed is digested to completion with a

restriction endonuclease

2 Electrophoresis to maximally separate restriction fragments

in the expected size range A set of standards of known size

is run in one lane of the gel

3 Blot fragments onto a nitrocellulose membrane

4 Hybridize with the 32P probe

5 Autoradiography

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Step 2

Gel electrophoresis

• Separates DNA fragments

Soak gel in 0.5 M NaOH

• Converts dsDNA to ssDNA

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Step 3 Nitrocellulose Blot

• Cover gel with nitrocellulose

paper…then…

• Cover nitrocellulose paper with

thick layer of paper towels

• Compress apparatus with heavy

weight

• ssDNA binds to nitrocellulose at

same position it had on the gel

• Vacum dry nitrocellulose at

80C to permanently fix DNA in

place or cross link (via covalent

bonds) the DNA to the

membrane

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Step 4 Hybridization

• Incubate nitrocellulose sheet with

a minimal quantity of solution

containing 32P-labeled ssDNA

temperature that will permit

probe to anneal to its target

sequence(s)

• Wash & dry nitrocellulose sheet

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Step 5 Autoradiography

• Place nitrocellulose sheet over

X-ray film

• X-ray film darkens where the

fragments are complementary to

the radioactive probes

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Northern Blots

1 Isolate RNA & treat with formaldehyde

2 Electrophorese RNA in denaturing agarose gel (has

formaldehyde) Visualize RNA in gel using Ethidium bromide stain and photograph

3 Transfer single-stranded RNA to nitrocellulose or nylon

membrane Covalently link RNA to membrane

4 Incubate membrane (RNA immobilized on membrane) with

labeled DNA or RNA probe with target sequence

5 Development

Total RNA on agarose gel

Northern Blot Results

Northern Blot Results (cont.)

When trying to learn about the function of a certain protein, it is

sometimes useful to purify mRNA from many different tissues or cell types and then prepare a Northern blot of those mRNAs, using a

cDNA clone of the protein of interest as the probe

Only mRNA from the cell types that are synthesizing the protein will hybridize to the probe

Example:

Expression sybII gene

at different life stages in the

frog Xenopus laevis

http://www.xenbase.org/WWW/Marker_pages /CNS/sybII.html

• No need to digest DNA

• Denature “folded” RNA with formaldehyde

• Probe with DNA or RNA

Western Blot encyclopedia

Western Blot Principle

Western blot is an important technique used in cell and molecular biology By using a western blot,

researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells The basic principle of western blot is to use three elements to accomplish this task:

(1) separation by size,

(2) transfer to a solid support, and

(3) marking target protein using a proper primary and secondary antibody to visualize

Blot type Target Probe Applications

Southern DNA DNA or

RNA

mapping genomic

clones estimating gene

PCR - Polymerase Chain Reaction

• PCR is an in vitro technique for the amplification of a region of DNA which lies

between two regions of known sequence

• PCR amplification is achieved by using oligonucleotide primers

– These are typically short, single stranded oligonucleotides which are

complementary to the outer regions of known sequence

• The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template

– This results in the synthesis of new DNA strands which are complementary

to the parent template strands

– These new strands have defined 5' ends (the 5' ends of the oligonucleotide primers), whereas the 3' ends are potentially ambiguous in length

Denaturing Template DNA

Heat causes DNA

The exact-length target

product is made in the

third cycle

The PCR

Reaction

How does it work?

Heat (94oC) to denature DNA strands

Cool (52oC) to anneal primers to template

Warm (72oC) to activate Taq polymerase, which extends

primers and replicates DNA

Repeat 35 cycles

The PCR cycle

(template) is separated into two stands

by heating to 95℃

reduced to around 55℃ to allow the

primers to anneal

to 72℃ for optimal polymerization step

which uses up dNTPs and required Mg++

• Primer is an oligonucleotide sequence, targets as complementary on single-stranded nucleic acids

DNA Polymerase

• DNA Polymerase is the enzyme responsible for copying the sequence starting at the primer from the single DNA strand

• Commonly use Taq, an enzyme from the

hyperthermophilic organisms Thermus aquaticus,

isolated first at a thermal spring in Yellowstone National Park

• This enzyme is heat-tolerant  useful both because it is thermally tolerant (survives the melting T of DNA

denaturation) which also means the process is more

specific, higher temps result in less mismatch – more

specific replication

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PCR Applications

• Amplification of DNA

• Modification of ends for cloning (RACE)

• Analysis of PCR products (nested primers)

• Cloning of genes (amplification from genome or library)

• Introduction of site-specific mutations

• Joining ends (religation of different DNA molecules) without ligation

• Invitro splicing

• Reverse Transcriptase (RT)-PCR

• Real-time PCR -> Diagnostics

• Asymmetric PCR -> ssDNA -> sequencing

• Detection of Infections (bacterial, viral) -> Diagnostics

• Detection of sex in prenatal cells

• Fingerprinting -> forensic medicine

• PCR on a Chip -> Detection of human pathogen organisms

• In situ PCR -> studying disease states, mapping chromosomes,…

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Adding of restriction sites for cloning of

a PCR product

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RT-PCR – Reverse Transcriptase PCR

First strand synthesis (single strain DNA)

Reverse transcriptase,

Primer (oligo(dT) or hexanucleotides or specific primer) and dNTPs

Second strand synthesis

best way of making full-length cDNA is to employs a ribonuclease (RNase H) which recognizes the RNA component of a DNA: RNA hybrid and cleaves the RNA at a number of non-specific sites

Ribonuclease H (RNase H) is an endonuclease that specifically hydrolyzes

the phosphodiester bonds of RNA hybridized to DNA to produce 3´-OH and 5´-P-terminated products It will not degrade single-stranded nucleic acids, double-stranded DNA or double-stranded RNA

Real-Time PCR or quantitative PCR

Exponential phase vs plateau

• At some time or another, all reactions

regardless of initial amount reach the same

plateau!

– Plateau is not quantitative

– Exponential phase is quantitative

Before the Real-Time era: End-point PCR

What is qPCR

• “quantitative Polymerase Chain Reaction”

• A method that allows to follow in real time

(that is why is also called Real-Time PCR) the amplification of a target

• The target can be nucleic acids (RNA or DNA)

• Taq polymerase can only synthesize DNA, so how do we study RNA using qPCR?

• Two common methods for the detection of

products in quantitative PCR are:

(1) non-specificfluorescent dyes that intercalate with any double-stranded DNA

(2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with

a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence to quantify messenger RNA (mRNA) and non-coding RNA in cells or tissues

Different chemistries involved to

obtain quantitative signal

TaqMan

Molecular beacons

Molecular probes

SYBRGreen

cDNA libraries

• The most commonly chosen genomic cloning vectors are λ replacement

vectors which must be digested with restriction enzymes to produce the two λ end fragment or λ arms between which the genomic DNA will be ligated

1 Characteristics of cDNA libraries

2 Methods to isolate mRNA

3 Check the mRNA integrity

4 Cloning the particular mRNAs

Characteristics of cDNA libraries

 No cDNA library was made from prokaryotic mRNA

• Prokaryotic mRNA is very unstable

• Genomic libraries of prokaryotes are easier to make and contain

all the genome sequences

 cDNA libraries are very useful for eukaryotic gene

analysis

• cDNAs represent the transcribed parts of the genome (i.e the

genes rather than the nontranscribed DNA) cDNAs have no

introns  genes can be expressed in E coli directly

• Tissue or cell type specific

cDNA libraries

mRNA isolation, purification

Check the RNA integrity

Treatment of cDNA ends

Blunt end ligation of large fragment is not efficient, so

we have to use special acid linkers to create sticky ends for cloning

The process

Move protruding 3’-ends (strand-special

nuclease)

Fill in missing 3’ nucleotide (klenow fragment of

DNA polyI and 4 dNTPs)

Ligate the blunt-end and linkers (T4 DNA ligase)

Restriction enzyme digestion (E.coRI )

Tailing with terminal transferase

or using adaptor molecules

Ligate vector and cDNA with T4 DNA ligase

(plasmid or λ phage vector)

Creating the cDNA library

Restriction enzyme

Creating the cDNA library

Pituitary genes

Restriction Enzyme

Pituitary genes

Restriction Enzyme

Creating the cDNA library

Pituitary genes

Restriction Enzyme

Transformation Host cells

Recombinant

Plasmid

Transformation

How to generate Genomic information

• There two ways in which

genomic information is

obtained

• Genomic library which

contains the entire human

Genome (exons and introns)

• cDNA (complementary

DNA) library the contains

only expressed genomic

information (only exons)

Genomic libraries — Genomic DNA

Purify genomic DNA

Correct size for cloning into the chosen vector:

Physical shearing and restriction enzyme digestion

Eukaryotes

Prokaryotes

Clone the fragments into vectors

Transformation into host cell

Restriction enzyme digestion

Partial digestion:

To get a greater lengths of DNA fragments

Time of digestion Ration of restriction enzyme to DNA

Partial Digestion

Break DNA into fragments randomly

Physical shearing

Pipeting, mixing or sonication The choice of method and time of exposure depend on the size requirement of the chosen vector

Ends produced (sticky or blunt) & the cleaved ends of the vector to be cloned

DNA modifications

Whether the enzyme is inhibited by DNA

modifications (CpG methylation in mammals)

replacement vectors which must be digested with restriction

which the genomic DNA will be digested

Genomic

library

Average Restriction Fragment Length

n = 4, 256 base pairs

n = 6, 4096 base pairs

n = 8, 65.5 kb base pairs

How many genomic clones must be

screened to find your gene?

Theoretically, you will need to screen N clones where

gene and f=the average size of the cloned genomic

sequence in your vector divided by the total genome

Genomic Sequences and Coverage

N = ln(1 - P)

ln(1 - f)

N = number of clones

P = probability of recovering a sequence,

f = fraction of the genome of each clone

E coli vs Humans

# Clones = ln(1 - P) ln(1 -

v/g)

P = probability of including any one sequence

v/g = insert size / genome size

n = 2900 mb / 20 kb insert = 145,000

P = 0.999

# Clones = 1,001,621

3 Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes

4 Infection and formation

Screening procedures

Screening Colony and plaque hybridization

Expression screening Hybrid arrest and release Chromosome walking (repeat screening)

Screening procedures

Colony and plaque hybridization

Transfer the DNA in the plaque or colony to a Nylon or nitrocellulose membrane

Phage DNA bind to

the membrane directly

Bacterial colonies must be lysed to release DNA on the membrane surface

Containing Nucleic acid probe)

(Alkali treatment)

Screening libraries

product

radioactively labeled or fluorescently labeled DNA or RNA

Direct Fluorescent- Labeled probe

Biotin; “Reporter group “: Alkaline phosphatase

and horseradish peroxidase

Chemiluminescence

Chemiluminescence: chemiluminescent

chemicals attached to the probe are detected by

their light emission using a luminometer

Fluorescence Chemicals: attached to probe

fluoresce under UV light-useful for the direct

examination of microbiological or cytological

specimens under the microscope – a technique

known as fluorescent in situ hybridization (FISH)

Antibodies

An antigenic group is coupled to the probe and its

presence detected using specific antibodies Also,

monoclonal antibodies have been developed that

will recognize DNA-RNA hybrids

Probe labels

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Colony Hybridisation

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Screening of Libraries

1 Hybridisation:

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Plaque Hybridisation

Secondary Antibody: against

proteins (antibodies) produced in

rabbit, mouse, bird,… (unspecific

but labeled)