recombinant dna technology; pc - wordpress.com · 2018-03-14 · tools for genetic engineering...

Recombinant DNA technology; PCR and its applications

Mitesh Shrestha

Genetic Engineering

• Genes are isolated, modified, and inserted into an organism

• Made possible by recombinant technology

– Cut DNA up and recombine pieces

– Amplify modified pieces

3

Recombinant DNA

• A DNA molecule consisting of two or more DNA segments that are not found together in nature.

• We can insert a gene into a plasmid, and infect a cell with the plasmid.

• “designer genes”

Tools for Genetic engineering

• Enzymes

• Vectors

• Host

Tools for Genetic engineering

Vectors • Vectors - small pieces of DNA used for cloning (the gene to be

inserted into the genetically modified organism must be combined

with other genetic elements in order for it to work properly)

• Requirements of the Vector 1. Self-replication - able to replicate in the host (origin of

repliction)

2. Cloning site (site for recognition of restriction nucleases)

3. Promoter (and operator) - to support the gene (new DNA) expression in the

host

4. Selectable marker – antibiotic resistance

5. Proper size

Vectors

1. Plasmid vectors

– Plasmids are self-replicating circular molecules of DNA

– Encode antibiotic resistance ( selection marker)

2. Viral vectors - retroviruses, adenoviruses and herpes viruses

– Accept much larger pieces of DNA

– Mammalian hosts

1. Bacteria

- E. coli - used because is easily grown and its

genomics are well understood.

– Gene product is purified from host cells

2. Yeasts - Saccharomyces cerevisiae

– Used because it is easily grown and its genomics are known

– May express eukaryotic genes easily

– Continuously secrete the gene product.

– Easily collected and purified

3. Plant cells and whole plants


– Plants are easily grown - produce plants with new properties.

4. Mammalian cells


– Harder to grow

– Medical use.

Hosts for DNA recombinant technology

Techniques for producing recombinant cells

1.Transformation

* treatment make cells competent to accept foreign DNA (CaCl2 make

pores in cell membrane)

2. Electroporation

*use electrical current to form microscopic pores in the membranes of

cell

3. Protoplast fusion

– yeast, plants and algal cells

4. Microinjection

5. Gene gun

Figure 9.5b

Recombinant DNA Cloning Procedure


1) Enzymatic digestion


2) Ligation of Target and vector

DNA Ligase


3) Transform Ligated DNA into Bacteria

Figure 9.1.2

An Overview of Recombinant DNA Technologies 1. Gene of interest (DNA) is isolated (DNA fragment)

2. A desired gene is inserted into a DNA molecule - vector

(plasmid, bacteriophage or a viral genome)

3. The vector inserts the DNA into a new cell, which is grown to form a clone.

(bacteria, yeast, plant or animal cell)

4. Large quantities of the gene product can be harvested from the clone.

In genetic engineering, recombination can also refer to artificial and deliberate recombination of pieces of DNA, from different organisms, creating what is called recombinant DNA.

Applications of recombinant DNA technology

1. Scientific applications

– Many copies of DNA can be produced

– Increase understanding of DNA

– Identify mutations in DNA

– Alter the phenotype of an organism

– Bioinformatics is the use of computer applications to study genetic data;

– Proteomics – proteomics is the study of a cell’s proteins. • determination of all the proteins expressing in the cell


• Shotgun sequencing - Recombinant DNA techniques were used to map the human genome through the Human Genome Project - has 24 distinct chromosomes (22 autosomal + X + Y)

- with a total of approximately 3 billion DNA base pairs

– containing an estimated 20,000–25,000 genes

– with only about 1.5-2% coding for proteins

– the rest comprised by RNA genes, regulatory

sequences, introns and controversially so-called

junk DNA

– This provides tools for diagnosis and

possibly the repair of genetic diseases

2. Recombinant DNA techniques can be used to for genetic

fingerprinting identification

• Forensic microbiology - use

DNA fingerprinting to identify

the source of bacterial or viral

pathogens.

– bioterrorism attacks (Anthrax in U.S. Mail)

– medical negligence (Tracing HIV to

a physician who injected it)

– outbreaks of foodborne diseases

Figure 9.16


4. Agricultural Applications

– Cells from plants with desirable characteristics can be cloned to

produce many identical cells, then can be used to produce whole plants

from which seeds can be harvested.

– Some bacteria can transfer genes to unrelated species

• Agrobacterium tumefaciens - a plant pathogen

• Cause tumors in plants

• Natural genetic engineer


Genetic engineering manipulation

GMO

Site of insertion foreign DNA

Selection

• Genes for resistance to herbicide glyphosate, Bt toxin, and

pectinase suppression have been engineered into crop plants.

• Genetically modified Rhizobium has enhanced nitrogen

fixation.

• Genetically modified Pseudomonas is a biological insecticide

that produces Bacillus thuringiensis toxin.

5. Nanotechnology

• Bacteria can make molecule-sized particles

– Bacillus cells growing on selenium form chains of elemental selenium


Applications of recombinant DNA technology in

Medicine

(1) Treatment of genetic diseases (gene

therapy)

• e.g. SCID girl

(2) Production of

medically useful

biologicals (e.g.

insulin)

Recombinant Human Growth Hormone

Recombinant insulin (Humulin)

(3) Vaccines production

• Firstly, the gene in a

pathogenic virus that

stimulates protective immunity

should be identified.

• That portion of DNA is then

isolated and incorporated into

an established harmless virus

(e.g. vaccinia virus).

• This new recombinant virus is used as a vaccine.

• These vaccines are much safer since they do not expose the patients to the actual virus and do not risk to infection.

• This method may be useful in vaccines against malaria and schistosomiasis and many viruses (e.g. HBV)

(4) Pharmacogenomics Deals with the influence

of genetic variation on drug response in patients by correlating gene expression with a drug's efficacy or toxicity

Design drugs adapted to an individual's genetic make-up

Polymerase Chain Reaction

PCR – first described in mid 1980’s, Mullis Nobel prize in

1993

An in vitro method for the enzymatic synthesis of

specific DNA sequences

Selective amplification of target DNA from a heterogeneous,

complex DNC/cDNA population

Requires

Two specific oligonucleotide primers

Thermostable DNA polymerase

dNTP’s

Template DNA

Sequential cycles of (generally) three steps (temperatures)

Required a thermostable DNA polymerase - Taq

DNA polymerase from Thermus aquaticus

a thermophilic eubacterial microorganism

isolated from a hot spring in Yellowstone

National Park

Kcat = 150 nucleotides/sec/enzyme (at Topt)

Taq1/2 =

Initially PCR used the Klenow fragment of E. coli DNA polymerase - inactivated by high temperatures Kleppe, Ohtsuka, Kleppe, Molineux, Khorana. 1971. J. Mol. Biol. 56:341.

5 min 97.5 oC

40 min 95.0 oC

130 min 92.5 oC

PCR - before the thermocycler

8 BORING hours per PCR!

95º C 5 min

35 times

55º C 3 min

72º C 5 min

heated lids

adjustable ramping times

single/multiple blocks

gradient thermocycler blocks

Thermocyclers

standard tube, volume, cost

evaporation & heat transfer

concerns

thin walled tube, volume, cost

evaporation & heat transfer

concerns

Directional Synthesis

“Xeroxing” DNA

1 copy

Cycle 35

n36 = 68,719,476,736 copies in ~ 2 hrs

2 copies

Cycle 2

4 copies

Cycle 3

8 copies

http://www.bst.ntu.edu.tw/index.htm

A simple thermocycling protocol

an

nealin

g

94ºC 94ºC

55ºC

72ºC

4ºC

3 min 1 min

45 sec

1 min

∞ hold

Initial denaturation

of DNA

1X 35X 1X

exte

nsio

n

den

atu

ratio

n

Step 1:

Denaturation

dsDNA to ssDNA

Step 2:

Annealing

Primers onto template

Step 3:

Extension

dNTPs extend 2nd strand

extension products in one cycle serve as template in the next

Basic Components of PCR

• Template DNA (0.5 - 50 ng) < 0.1 ng plasmid DNA, 50 ng to 1 μg gDNA for single copy genes

• Oligonucleotide primers (0.1 – 2.0 μM)

• dNTP’s (20 –250 μM)

• Thermostable DNA pol (0.5 – 2.5 U/rxn)

• MgCl2 (1 – 5 mM) affects primer annealing and Taq activity

• Buffer (usually supplied as 10X) Working concentrations

KCL (10 – 50 mM)

Tris-HCl (10 mM, pH 8.3)

NaCl2 (sometimes)

dNTPs Taq polymerase

Primers

DNA template

Buffer

+ +

A C T G

MgCl2

Magnesium Chloride

(MgCl2 - usually 0.5-5.0mM)

Magnesium ions have a variety of effects

Mg2+ acts as cofactor for Taq polymerase

Required for Taq to function

Mg2+ binds DNA - affects primer/template interactions

Mg2+ influences the ability of Taq pol to interact with

primer/template sequences

More magnesium leads to less stringency in

binding

MgCl2 (mM)

1.5 2 3 4 5

PCR Problems

Taq is active at low temperatures

At low temperatures mis-priming is likely

Extension Rate Temp

0.25 nt/sec 22o C

1.5 nt/sec 37o C

24 nt/sec 55o C

150 nucleotides in 10 min

• “Cheap” fixes

– Physical separation –”DNA-in-the-cap”

– Set up reactions on ice

• Hot-start PCR –holding one or more of the PCR components until the first heat denaturation

– Manually - delay adding polymerase

– Wax beads

– Polymerase antibodies

• Touch-down PCR – set stringency of initial annealing temperature high, incrementally lower with continued cycling

• PCR additives

– 0.5% Tween 20

– 5% polyethylene glycol 400

– betaine

– DMSO

“Cures” for mis-priming

Primer Design

1. Typically 20 to 30 bases in length

2. Annealing temperature dependent upon

primer sequence (~ 50% GC content)

3. Avoid secondary structure, particularly 3’

4. Avoid primer complementarity (primer dimer)

5. The last 3 nucleotides at the 3` end is the

substrate for DNA polymerase - G or C

6. Many good freeware programs available

Primer Dimers

• Pair of Primers

5’-ACGGATACGTTACGCTGAT-3’

5’-TCCAGATGTACCTTATCAG-3’

• Complementarity of primer 3’ ends

5’-ACGGATACGTTACGCTGAT-3’

3’-GACTATTCCATGTAGACCT-5’

• Results in PCR product

Primer 1

5’-ACGGATACGTTACGCTGATAAGGTACATCTGGA-3’

3’-TGCCTATGCAATGCGACTATTCCATGTAGACCT-5’

Primer 2

Rules of thumb for PCR conditions

• Add an extra 3-5 minute (longer for Hot-start Taq) to your cycle

profile to ensure everything is denatured prior to starting the PCR

reaction

• Approximate melting temperature (Tm) = [(2 x (A+T)) +(4 x

(G+C))]ºC

• If GC content is < 50% start 5ºC beneath Tm for annealing

temperature

• If GC content ≥ 50% start at Tm for annealing temperature

• Extension @ 72ºC: rule of thumb is ~500 nucleotide per minute.

Use 3 minutes as an upper limit without special enzymes

• “Special” PCR cycling protocols

• Touchdown PCR

• Step-up PCR

• Gradient cycling

Common PCR additives

BSA (usually at 0.1 to 0.8 µg/µL final concentration)

Stabilize Taq polymerase & overcome PCR inhibitors

DMSO (usually at 2-5% v/v, inhibitory at ≤ 10% v/v)

Denaturant - good at keeping GC rich template/primer strands

from forming secondary structures.

Glycerol (usually at 5-10% v/v)

Increases apparent concentration of primer/template mix, and

often increases PCR efficiency at high temperatures.

Stringency enhancers (Formamide, Betaine, TMAC)

Concentrations used vary by type

Enhances yield and reduces non-specific priming

Non-ionic detergents (Triton X, Tween 20 or Nonidet P-40) (0.1–1%)

NOT SDS (0.01% SDS cuts Taq activity to ~10% of normal)

Stabilize Taq polymerase & suppress formation of 2º structure

PCR additives - Literature

Additive References

DMSO (dimethyl sulfoxide)

Amplifications 5: 16

Gene 140: 1

Nucleic Acids Research 18: 1666

Betaine (N,N,N-trimethylglycine

= [carboxymethyl]

trimethylammonium)

Biochemistry 32: 137

BioTechniques 21: 1102

Genome Research 6: 633


Proceedings of the National Academy of Sciences of the United States of America

70: 298

Trends in Biochemical Science 22: 225

Formamide Nucleic Acids Research 18: 7465

Non-ionic detergents e.g. Triton X-100, Tween 20 or

Nonidet P-40 (NP-40)

Biotechniques 12: 332


TMAC (tetramethylammonium chloride)



dC7GTP (7-deaza-2'-deoxyguanosine)


BSA (bovine serum albumin)

Applied and environmental microbiology 62:1102-1106

BioTechniques 23:504

BioTechniques 25:564


Typical PCR Temps/Times

hold 4o C or 10 mM

EDTA

Stop reaction

5 – 10

min

70o – 75o C Final

extension

0.5 – 2

min

70o – 75o C Primer

extension

0.5 – 1

min

45o – 65o C Primer

annealing

0.5 – 1

min

90o – 95o C Denature

1 – 3

min

90o – 95o C Initial

denaturation

25 – 40

cycles

Troubleshooting PCR

Non-specific bands on your gel

Reagents, set-up Run negative control

Template concentration inappropriate Review guidelines

Annealing temp too low Optimize by gradient PCR

Extension time too short time for longer products

Cycle number too high Review guidelines

Primer design not appropriate specificity

Primer concentration too high Optimize by titration

Non-specific priming specificity, Hot Start

MgCl2 concentration too high Optimize by titration

GC-rich template, 2° structure PCR additives

Contaminating DNA Decontaminate work area:

use ARTs, wear gloves,

pipettor, reagents,

UV treat plastics

Troubleshooting PCR

Diffuse smearing on your gel

Template concentration inappropriate Review guidelines

Taq concentration too high Optimize by titration

Extension time inappropriate Review guidelines

Cycle number too high Reduce, review guidelines

Primer design not appropriate specificity


Non-specific priming use Hot Start

MgCl2 concentration too high Optimize by titration


Contaminating DNA Decontaminate work area:

use ARTs, wear gloves,

pipettor, reagents,

UV treat plastics

Troubleshooting PCR

Poor or no amplification of bands

Problem with thermocycler, set-up, Run positive control

reagents

Enzyme concentration low Concentration

Annealing temp too low Optimize by gradient PCR

Extension time too short Time for longer products

Cycle number too low Review guidelines

Primer design not appropriate Specificity


Non-specific priming Specificity, Hot Start

MgCl2 concentration too low Optimize by titration


Troubleshooting PCR

Prioritizing Approaches

“Pilot” error ( set-up errors common in the interim between

training with someone and working independently)

Template dilution error (concentration matters!)

Thermocycling parameter errors (temps/times)

Bad reagents (1. dNTPs, 2. primers, 3. Taq)

Unique template or template structure issues

Don’t get discouraged…validating PCRs can be tricky

Applications of PCR

• Classification

of organisms

• Genotyping

• Molecular

archaeology

• Mutagenesis

• Mutation

detection

• Sequencing

• Cancer research

• Detection of

pathogens

• DNA

fingerprinting

• Drug discovery

• Genetic

matching

• Genetic

engineering

• Pre-natal

diagnosis

Applications of PCR

Basic Research Applied Research

• Genetic matching • Detection of pathogens • Pre-natal diagnosis • DNA fingerprinting • Gene therapy

• Mutation screening • Drug discovery • Classification of organisms • Genotyping • Molecular Archaeology • Molecular Epidemiology • Molecular Ecology

• Bioinformatics

• Genomic cloning

• Site-directed mutagenesis

• Gene expression studies

Applications of PCR

Molecular Identification Sequencing Genetic Engineering

• Molecular Archaeology • Molecular Epidemiology • Molecular Ecology • DNA fingerprinting • Classification of organisms • Genotyping • Pre-natal diagnosis • Mutation screening • Drug discovery • Genetic matching • Detection of pathogens

• Bioinformatics

• Genomic cloning

• Human Genome Project

• Site-directed mutagenesis

• Gene expression studies

recombinant dna technology; pc - wordpress.com · 2018-03-14 · tools for genetic engineering...

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