8.1 Recombinant DNA technology
2
8.2 Methods in Gene Cloning
2
8.3 Application of Recombinant DNA Technology 1
(a) Define recombinant DNA technology (b) State the tools used in recombinant DNA technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends (d) List and explain the types of cloning vectors (e) Describe the characteristics of cloning vector (f) Describe host cell (bacteria) and its characteristics (g) Define modifying enzymes and its function
Learning outcomes 8.1 Recombinant DNA Technology a) define recombinant DNA technology
Recombinant DNA technology Gene Technology : techniques and methods used in understanding gene expression and gene manipulation. Gene manipulation/genetic engineering : modifying and recombining DNAs to produce desired products such as proteins, or animals and plants with desirable traits.
Learning outcomes 8.1 Recombinant DNA Technology a) define recombinant DNA technology
Define recombinant DNA technology Manipulating the information content of DNA through the use of recombinant DNA (rDNA) rDNA; DNA in which genes from 2 different sources are linked
Learning outcomes 8.1 Recombinant DNA Technology (b) State the tools used in recombinant DNA technology
Tools used in recombinant DNA technology i. Target DNA (gene of interest) – the gene to be cloned ii. Restriction enzymes – to cut DNA into fragments iii. DNA cloning vector – to carry the target gene into a host cell. Example: plasmid and bacteriophage iv. Host cell – bacterial cells that allows the cloning vector to replicate within it v. Modifying enzymes – to join DNA fragments together. Example: DNA ligase
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Restriction enzyme Restriction enzyme are naturally found in certain
bacteria that are resistance to bacteriophage infection. Different restriction enzymes cut at a specific restriction site to produce DNA fragments with sticky ends or blunt ends Restriction enzyme has an ability to cut through DNA at specific sequence
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Restriction site The restriction site is a DNA sequence that
consists of the same four to eight nucleotides on both strands but arranged in opposite direction. 5’–AAGCTT–3’reads 3’–TTCGAA–5’
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Restriction site : Palindrome sequence Palindrome: the base sequence of one strand
which reads the same as its complement, but in the opposite direction on the double stranded DNA. 5’–AAGCTT–3’reads 3’–TTCGAA–5’ The recognition site for restriction enzyme To cut/restrict both DNA strand
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Restriction enzyme • Two main types of restriction enzymes: 1. cut DNA fragments forming ‘sticky ends’ Produced when the restriction enzyme produce staggered cut Hanging complimentary single strands of DNA at the cut ends. DNA with complimentary sticky ends are able to anneal/join
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Restriction enzyme
• Two main types of restriction enzyme: 2. cut DNA fragments forming ‘blunt ends’
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Examples of enzymes that produce sticky ends and blunt ends
Enzyme
Produced by organism
1. EcoRI
Escherichia coli
Restriction sequence G A A T T C C T T A A G
2. BamHI Bacillus amyloquefacein Serrana marcescens
Sticky end
G G A T C C C C T A G G
3. SmaI
Sticky or blunt end
Sticky end
C C C G G G G G G C C C
Blunt end
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Examples of enzymes that produce sticky ends
5’
3’
G A A T T C
C T T A A G
3’
5’
a) Restriction enzyme, EcoRI- Recognize specific nucleotide sequences GAATTC, and cut the DNA. Sequences recognized are palindromes
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Examples of enzymes that produce sticky ends
C C G A A T T C G G G G C T T A A G C C b) EcoRI cuts a double stranded DNA & produces DNA fragments with STICKY ENDS TTAA and AATT
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Examples of enzymes that produce sticky ends
A A T T C G G C C G G G C T T A A c) 2 DNA fragments with sticky ends ( no complementary bases) are formed
G C C
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Examples of enzymes that produce blunt ends
5’
3’
C C C G G G 3’
G G G C C C
5’
a) Restriction enzyme, SmaI- Recognize specific nucleotide sequences CCCGGG, and cut the DNA.
Sequences recognized are palindromes
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Examples of enzymes that produce blunt ends
b) Sma I cuts the phosphodiester bond between the third and the fourth nucleotide in the recognition sequence (containing nitrogenous bases C and G) in the upper strand. It also cuts at the same sequence in the strand below (read the sequence of DNA from 5’ to 3’). Since it cuts at the same places in both strand, the two strands is completely cut.
Learning outcomes 8.1 Recombinant DNA Technology (c) Explain restriction enzyme and examples of enzymes that produce sticky ends and blunt ends
Examples of enzymes that produce blunt ends
c) 2 DNA fragments with blunt ends ( no complementary bases) are formed
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
List and explain the types of cloning vectors
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
Cloning vectors • Cloning Vector – an agent / DNA molecule derived from plasmid/bacteriophage/cosmid/YAC that carries foreign DNA fragment into a host cell.
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
Cloning vectors • The foreign DNA is inserted into a cloning vector which then replicates in the host cell. • Types; plasmid, bacteriophage, cosmid, YAC (Yeast artificial chromosome)
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
Types of DNA vector i. Plasmids ( eg : pUC 18) small, circular, double stranded DNA molecules found in certain bacteria. carry few genes such as genes coding for resistance to antibiotics They are separated from the larger portion of the DNA Can carry 10 – 15 thousand base pairs ( 10 – 15 kb) Can replicate independently of the rest of the DNA
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
Types of DNA vector i. Plasmids ( eg : pUC 18) Characteristics of plasmid (PSPM 2005/2006) - contain origin of replication - plasmid replicates independently - contains multiple cloning site - allows much more diverse DNA fragment to be inserted - contain selectable marker / ampR - resistant to antibiotic - allows the cells that contaion the recombinant DNA to be readily screened - ability to express cloned genes
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
Types of DNA vector Bacterial plasmids ("Blue Genes") X110,000.
plasmids pSC101
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
Types of DNA vector ii. Bacteriophages (eg: λ 2001) are viruses which can insert their rDNA into bacteria for replication More efficient in transformation compared to plasmid Can carry up to 25 kb of foreign DNA
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
Types of DNA vector ii. Bacteriophages (eg: λ 2001)
Phage DNA can accept foreign DNA restriction fragment (forming recombinant DNA) Phage infects E. coli (by injecting its recombinant DNA) Recombinant DNA replicates in E. coli (producing recombinant DNA copies)
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
Types of DNA vector iii. Cosmid ( eg sCOS-1) A hybrid of the plasmid and the phage. It can then infect the host cell in a phage-like manner. Can carry up to 45 kb
Learning outcomes 8.1 Recombinant DNA Technology (d) List and explain the types of cloning vectors
Types of DNA vector iv. YAC (Yeast artificial chromosome) – eg pYAC Artificial DNA molecules that carry out DNA replication and gene expression in yeast cell capable of carrying a large DNA fragments up to 2000 kb Transformation efficiency is low
Learning outcomes 8.1 Recombinant DNA Technology (e) Describe the characteristics of cloning vector
(e) Describe the characteristics of cloning vector
Learning outcomes 8.1 Recombinant DNA Technology (e) Describe the characteristics of cloning vector
Characteristics of DNA vector
i. Have an ability to accept foreign DNA fragment ii. Have an origin of replication initiation – ori, able to replicate freely in the host cell
Learning outcomes 8.1 Recombinant DNA Technology (e) Describe the characteristics of cloning vector
Characteristics of DNA vector
iii. Have a selectable marker genes for screening iv. Have an ability to express cloned genes v. Cloned gene can be recovered
Learning outcomes 8.1 Recombinant DNA Technology (e) Describe the characteristics of cloning vector
Characteristics of DNA vector
vi. Able to transfer foreign gene to the host cell vii. Having a multiple cloning site/polycloning site- contain several enzymes recognition sites - allows much more diverse DNA fragment to be inserted
Learning outcomes 8.1 Recombinant DNA Technology (f) Describe host cell (bacteria) and its characteristics
Describe host cell (bacteria) and its characteristics
Learning outcomes 8.1 Recombinant DNA Technology (f) Describe host cell (bacteria) and its characteristics
Host cell In order to
replicate, the cloning vectors carrying the target gene (recombinant DNA) must be introduced into the host cell.
Learning outcomes 8.1 Recombinant DNA Technology (f) Describe host cell (bacteria) and its characteristics
Host cell organism
that had been used to receive a recombinant DNA for cloning purposes bacteria (e.g : E. coli) and yeast.
Learning outcomes 8.1 Recombinant DNA Technology (f) Describe host cell (bacteria) and its characteristics
Characteristics of Host cell a) Able to receive/accept recombinant DNA through the transformation process b) Able to maintain the structure of the recombinant DNA from one generation to another c) Able to amplify the gene product from recombinant DNA d) Able to express the cloned gene
Learning outcomes 8.1 Recombinant DNA Technology (g) Define modifying enzymes and its function
Define modifying enzymes and its function
Learning outcomes 8.1 Recombinant DNA Technology (g) Define modifying enzymes and its function
Modifying enzymes - Enzyme used in modifying the DNA strands i. •
•
DNA ligase Joins 2 separate DNA fragments. It catalyses the formation of the phosphodiester bond (covalent bond). During the insertion of a target gene into a cloning vector, DNA ligase joins the target gene and plasmid to produce recombinant DNA
Learning outcomes 8.1 Recombinant DNA Technology (g) Define modifying enzymes and its function
Modifying enzymes - Enzyme used in modifying the DNA strands Taq polymerase thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus. Abbreviated to "Taq Pol" (or simply "Taq"), and is frequently used in polymerase chain reaction (PCR) ii.
8.1 Recombinant DNA technology
2
8.2 Methods in Gene Cloning
2
8.3 Application of Recombinant DNA Technology 1
(a) Describe the steps in gene cloning by using plasmid as a vector (b) Explain PCR (Polymerase Chain Reaction)
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
GENE CLONING • Process to produce multiple copies of certain genes/DNA fragments • Usually involves plasmids (as vectors) and bacteria (as host cells)
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
Steps in gene cloning by using plasmid i.
Isolation of gene
ii.
Cleave / cut
iii.
Insertion
iv.
Transformation and amplification
v.
Screening
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
The Steps in Cloning 1.
Isolation of vector and gene-source DNA.
2.
Splicing of donor and plasmid DNA with the same restriction enzymes.
3.
Insertion of DNA into the vector.
4.
Transformation of the bacterial cell (reintroduction of recombinant DNA into plasmid) Screening the desired cloned genes from the cloned cells.
5.
47
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
The Steps in Cloning
plasmid
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
1. Isolation
i.e. the extraction of the plasmid DNA of vector (e.g. E. coli) and the donor DNA (e.g. human).
♫
the plasmid DNA here carries two genes ampR and lacZ
♫
the restriction site is inside the lacZ 49
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
1. Isolation: Basic Procedures to release DNA: * the cell wall, the plasma membrane and the nuclear envelope are broken down by detergents and enzymes
to purify the DNA: * filtration @ centrifugation; i.e. to separate * protease and phenol are added
to precipitate the DNA: * ice-cool ethanol are poured in 50
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
2. Cleave/cut/digest 2. Splicing
the plasmid DNA of vector and the donor DNA are cut by the same RE at the restriction sites of the same palindromic base sequence producing thousands of donor DNA fragments and cut DNA plasmids
51
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
3. Insertion
after mixing, the DNA fragments and the cut plasmids form the complementary pairs
they are then joined by DNA ligase creating a mixture of recombinant DNA molecules and nonrecombinant plasmids
52
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
3. Insertion ♫
note that the lacZ has become nonfunctional
♫
i.e. cannot code for β-galactosidase
53
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
4. Transformation
the recombinat DNA (@ genetically engineered plasmids) are reintroduced into the bacteria
bacterial cells are mixed with recombinant DNA in the presence of cold calcium chloride
followed by heating; making the bacterial cell wall permeable to plasmids
54
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
DNA from donor
plasmid
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
4. Transformation
‘transforming’ the bacteria into the lacZ– bacteria, unable to hydrolyse lactose (mutation on their own LacZ gene) ♫
57
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
4. Transformation
produces diverse pool of bacteria
bacteria with desired recombinant DNA
bacteria with other recombinant DNA (wrong molecules)
bacteria with non-recombinant DNA (no plasmid)
bacteria with self-ligated vector
58
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector 2
Plasmid
3
Clone 1 Recombinant DNA and plasmids
+ Fragments
Cleaved plasmid
Clone 2
+
Recombinant DNA
Bacterial cells
Clone 3
Clone 4
Eliminate cells without plasmid
3
Clone 1 Recombinant DNA and plasmids
Clone 2
+
Bacterial cells
Clone 3
Clone 4
Ampicillin in media
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
5. Screening Procedure 1 medium containing antibiotic i.e. detecting the bacteria containing rDNA
♫
the colonies of bacteria is cultured on the solid medium containing the respective antibiotic
in this example, it is ampicillin
63
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
5. Screening The Observation
♫
only bacteria with ampicillin-resistant plasmid grow
64
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
5. Screening The Observation (continue….)
♫ Because…. Will resist the antibiotic and survive ♫But…. The bacteria that have not taken up plasmid, will die. ♫Because…. They are not resistant to the antibiotic. 65
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
17.3 Cloning
5. Screening 3
Clone 1 Recombinant DNA and plasmids
Clone 2
+
Bacterial cells
Clone 3
Clone 4
Eliminate cells without plasmid
Ampicillin in media
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
17.3 Cloning
5. Screening
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
5. Screening Procedure 2 Blue-white screening
♫
the X-gal is used to identify plasmid that carries foreign DNA
n
bacteria without foreign DNA stain blue as the β-galactosidase hydrolyses X-gal
n
bacteria with foreign DNA are white as they lack the β-galactosidase
• White colonies show bacterial clones which carry recombinant DNA clones. 68
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
5. Screening
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
5. Screening 3
Clone 1 Recombinant DNA and plasmids
Clone 2
+
Identify cells without rDNA Bacterial cells
Clone 3
X- gal in media
Clone 4
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector APPLICATION Cloning is used to prepare many copies of a gene of interest for use in sequencing the gene, in producing its encoded protein, in gene therapy, or in basic research. TECHNIQUE
In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes -galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (Xgal) to form a blue product. Only three plasmids and three human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples. lacZ gene Human (lactose breakdown)cell
Bacterial cell
1 Isolate plasmid DNA and human DNA. Restriction site R 2 Cut both DNA samples with the same restriction amp gene Bacterial (ampicillin plasmid enzyme resistance)
3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids.
Figure 20.4
Gene of interest Sticky ends
Recombinant DNA plasmids
Human DNA fragments
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
4 Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene.
5 Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow.
Recombinant bacteria
Colony carrying nonrecombinant plasmid with intact lacZ gene
Colony carrying recombinant plasmid with disrupted lacZ gene
Bacterial clone
RESULTS Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest.
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector Slide number: 1 Source DNA
Site
Site
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09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector Slide number: 1
Enzyme Source DNA
Site
Site
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09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector Slide number: 1
Enzyme Source DNA
Site
Site
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
4
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector Slide number: 1
Enzyme Source DNA
Site
Site
Gene of interest +
+ Fragments
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector Slide number: 1
Enzyme Source DNA
Site
Site
Gene of interest +
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
6
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector Slide number: 1
2
Enzyme
Plasmid
Source DNA
Site
Site
Gene of interest +
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
7
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector Slide number: 1
2
Enzyme
Plasmid
Source DNA
Site
Site
Gene of interest +
+ Fragments
Cleaved plasmid
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
8
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector Slide number: 1
2
Enzyme
Plasmid
Source DNA
Site
Site
Gene of interest +
+ Fragments
Cleaved plasmid
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
9
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaved plasmid
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
10
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaved plasmid
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
11
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaved plasmid
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Recombinant DNA
12
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Cleaved plasmid
Recombinant DNA
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
13
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Cleaved plasmid
Recombinant DNA
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
3
Recombinant DNA and plasmids
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
14
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Cleaved plasmid
Recombinant DNA
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
3
Recombinant DNA and plasmids +
Bacterial cells
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
15
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Cleaved plasmid
Recombinant DNA
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
3
Recombinant DNA and plasmids +
Bacterial cells
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
16
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Cleaved plasmid
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
3
Recombinant DNA and plasmids +
Recombinant DNA
Clone 1
Bacterial cells
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Cleaved plasmid
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
3
Recombinant DNA and plasmids +
Recombinant DNA
Clone 1 Clone 2
Bacterial cells
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Cleaved plasmid
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
3
Recombinant DNA and plasmids +
Bacterial cells
Recombinant DNA
Clone 1 Clone 2
Clone 4
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
19
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Cleaved plasmid
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
3
Recombinant DNA and plasmids +
Bacterial cells
Clone 1 Clone 2
Clone 3
Recombinant DNA
Clone 4
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
20
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
Cleaved plasmid
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
3
Recombinant DNA and plasmids +
Bacterial cells
Clone 1 Clone 2
Clone 3
Recombinant DNA
Clone 4
Cloning. A variety of recombinant plasmids are produced, some containing the gene of interest (red), others containing other fragments from the source DNA (blue), and still others containing no fragment. All are allowed to infect bacterial cells. Each cell reproduces and forms a clone of bacterial cells, each clone containing one type of plasmid. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Cleaved plasmid
Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
3
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
4
Recombinant DNA and plasmids +
Bacterial cells
Clone 1 Clone 2
Clone 3
Recombinant DNA
Clone 4
Cloning. A variety of recombinant plasmids are produced, some containing the gene of interest (red), others containing other fragments from the source DNA (blue), and still others containing no fragment. All are allowed to infect bacterial cells. Each cell reproduces and forms a clone of bacterial cells, each clone containing one type of plasmid. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
22
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Cleaved plasmid
Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
3
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
4
Recombinant DNA and plasmids +
Bacterial cells
Eliminate cells without plasmid.
Clone 1 Clone 2
Clone 3
Recombinant DNA
Clone 4
Cloning. A variety of recombinant plasmids are produced, some containing the gene of interest (red), others containing other fragments from the source DNA (blue), and still others containing no fragment. All are allowed to infect bacterial cells. Each cell reproduces and forms a clone of bacterial cells, each clone containing one type of plasmid. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
23
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Cleaved plasmid
Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
3
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
4
Recombinant DNA and plasmids +
Bacterial cells
Eliminate cells without plasmid.
Clone 1 Clone 2
Clone 3
Recombinant DNA
(Treat with antibiotic)
Clone 4
Cloning. A variety of recombinant plasmids are produced, some containing the gene of interest (red), others containing other fragments from the source DNA (blue), and still others containing no fragment. All are allowed to infect bacterial cells. Each cell reproduces and forms a clone of bacterial cells, each clone containing one type of plasmid. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
24
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+ Cleaved plasmid
Fragments
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
3
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
4
Recombinant DNA and plasmids +
Bacterial cells
Eliminate cells without plasmid.
Clone 1 Clone 2
Clone 3
Recombinant DNA
(Treat with antibiotic)
Clone 4
Cloning. A variety of recombinant plasmids are produced, some containing the gene of interest (red), others containing other fragments from the source DNA (blue), and still others containing no fragment. All are allowed to infect bacterial cells. Each cell reproduces and forms a clone of bacterial cells, each clone containing one type of plasmid. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
25
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
3
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
4
Recombinant DNA and plasmids +
Bacterial cells
Recombinant DNA
Cleaved plasmid
Fragments
Eliminate cells without plasmid.
Eliminate cells without recombinant DNA.
Clone 1 Clone 2
Clone 3
(Treat with antibiotic)
Clone 4
Cloning. A variety of recombinant plasmids are produced, some containing the gene of interest (red), others containing other fragments from the source DNA (blue), and still others containing no fragment. All are allowed to infect bacterial cells. Each cell reproduces and forms a clone of bacterial cells, each clone containing one type of plasmid. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
26
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
3
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
4
Recombinant DNA and plasmids +
Bacterial cells
Recombinant DNA
Cleaved plasmid
Fragments
Eliminate cells without plasmid.
Eliminate cells without recombinant DNA.
Clone 1 Clone 2
Clone 3
(Treat with antibiotic)
Clone 4
Cloning. A variety of recombinant plasmids are produced, some containing the gene of interest (red), others containing other fragments from the source DNA (blue), and still others containing no fragment. All are allowed to infect bacterial cells. Each cell reproduces and forms a clone of bacterial cells, each clone containing one type of plasmid. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
27
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
3
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
4
Recombinant DNA and plasmids +
Bacterial cells
Recombinant DNA
Cleaved plasmid
Fragments
Eliminate cells without plasmid.
Eliminate cells without recombinant DNA.
Clone 1 Clone 2
Clone 3
(Treat with antibiotic)
Find gene of interest
Clone 4
Cloning. A variety of recombinant plasmids are produced, some containing the gene of interest (red), others containing other fragments from the source DNA (blue), and still others containing no fragment. All are allowed to infect bacterial cells. Each cell reproduces and forms a clone of bacterial cells, each clone containing one type of plasmid. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
28
09_03 How a genetic engineering experiment works Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as number: the vector Slide 1
2
Enzyme
Plasmid
Source DNA
+ Site
Site
Gene of interest +
Fragments
+
Cleaving DNA. Enzymes cut the source DNA at specific sites, cleaving the two strands short distances apart.
3
Producing recombinant DNA. A circular plasmid cut with the same enzyme is combined with the fragments of source DNA.
4
Recombinant DNA and plasmids +
Recombinant DNA
Cleaved plasmid
Fragments
Eliminate cells without plasmid.
Eliminate cells without recombinant DNA.
Clone 1 Clone 2
(Treat with antibiotic)
Find gene of interest Yes
Bacterial cells
Clone 3
Clone 4
Cloning. A variety of recombinant plasmids are produced, some containing the gene of interest (red), others containing other fragments from the source DNA (blue), and still others containing no fragment. All are allowed to infect bacterial cells. Each cell reproduces and forms a clone of bacterial cells, each clone containing one type of plasmid. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
29
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
Cloning (PSPM 03/04) Discuss the process of producing human insulin through DNA recombinant technology using Escherichia coli
DNA containing the insulin gene is isolated. DNA is spliced using restriction enzyme. To form DNA fragments With sticky ends Vector/plasmid from E. coli is isolated The vector is cut (same RE)/complementary ends Plasmid opens Plasmid and human gene fragment bear complementary sticky ends To produce linear plasmid with sticky ends 101
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
Cloning (PSPM 03/04) Discuss the process of producing human insulin through DNA recombinant technology using Escherichia coli (continue..)
cDNA inserted into vector With aid of DNA ligase To form recombinant DNA/plasmid Recombinant DNA is transferred to host cell Transformation process Then screening is performed For cloning And amplification Insulin gene will be expressed Insulin is extracted from E.coli 102
Learning outcomes 8.2 Methods in Gene Cloning a) describe the steps in gene cloning by using plasmid as the vector
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
POLYMERASE CHAIN REACTION
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
Objectives By the end of the lesson the students should be able to: (a)Describe the steps in gene cloning by using plasmid as a vector (b) Explain about the Polymerase Chain Reaction 105
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
17.2 (f) Polymerase Chain Reaction Cloning the Genes: PCR A technique to rapidly replicate or amplify selected DNA segments that are initially present in extremely small amounts. In only few hours PCR can make millions or even billions of copies of particular DNA sequence
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
Cloning the Genes: PCR This technique will produce enough DNA material for DNA fingerprinting process Helps in (murder)
solving
of
violent
crimes
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
Cloning the Genes: PCR •
Polymerase Chain Reaction
•
for preparing large quantities of a particular gene in vitro
•
i.e. when the source of DNA is scanty or impure quicker and more selective can amplify any piece of DNA without using cells using DNA polymerase
• •
•
108
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
PCR: Procedures •
Incubation
(i) DNA is incubated in a test tube with special DNA polymerase(Taq polymerase) (iii) a supply of nucleotides (iv) primers in the form of single-stranded DNA
109
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
PCR: Procedures •
Three stages in PCR
1. 2. 3.
Denaturation Annealing Strand elongation/polymerization
110
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
PCR: Procedures Denaturation • •
i.e. brief heating at 90°C to separate DNA strands
111
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
PCR: Procedures Annealing
• • •
cooling/lowered temperature to 50°C to allow primers to bind to the template i.e. hydrogen bond
112
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
PCR: Procedures Polymerization/Extension • Heat resistant DNA polymerase (Taq Polymerase) adds nucleotides to the 3’ ends of each primer • until two molecules of DNA are yielded Cycle 1 completes 5’
•
the cycle repeats
3’
3’ 5’ 113
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
PCR: Procedures Polymerization/Extension •
Cycle 2 produces four DNA molecules
•
Cycle 3 produces eight
114
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
Polymerase Chain Reaction PCR: Review •Usually there are 30 cycles to complete to get sufficient amount of DNA
115
Learning outcomes 8.2 Methods in Gene Cloning b) explain the PCR (Polymerase Chain Reaction)
PSPM (07/08)
• PCR is an in-vitro process. • Involves a three step process. • Fisrt step is denaturation/separation of double-stranded DNA •By heating at high temperature (94-980C) • Second step is annealing of the primer to the target region • By cooling/lowering the temperature (50-650C). • Third step is strand elongation/polymerization/extension. • By Primer extension • At 70-720C • Catalyzed by DNA polymerase/Taq polymerase. • By adding free nucleotide. • in the 5’ to 3’ direction. • The cycle is repeated many times / 30-40 cycles. 116
8.1 Recombinant DNA technology
2
8.2 Methods in Gene Cloning
2
8.3 Application of Recombinant DNA Technology 1
Briefly explain and give examples of recombinant DNA Technology applications such as in; i. agriculture ii. Production of insulin iii. environment
Regular vs GOLDEN rice
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Definition : transgenic organisms • an organism whose genome contains and expresses genes from another species using recombinant DNA technology • a transgenic organism has desirable and improved characteristics (faster growth, larger muscle) •Combination of different genes and DNA sources are usually made in vitro
123
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
ETHICS IN GENETIC ENGINEERING • Genetically engineered organisms may possess danger to human or ecologically harmful to environment • animal rights are violated by treating animals as human properties • Might develop painful side-effect to the animals by creating ‘genetically superior’ characteristic
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Examples of rDNA technology applications
§
i. agriculture
§
ii. Production of insulin
§
iii. environment
125
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
i. agriculture Examples Animals
§ Transgenic salmon Plants/Crops
§
soybean resistant to herbicides 126
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Examples:
Resistance to herbicide
Transgenic plant: soybean resistant to herbicide
Insert genes encoding for proteins making crops resistant to herbicide Transgenic plants are plants that have been genetically engineered, a breeding approach that uses recombinant DNA techniques to create plants with new characteristics They are identified as a class of genetically modified organism (GMO) 127
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Inserting Genes into Plant Cells
Plant with gene for breaking down luciferin from fireflies
)
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Examples
Plants/Crops
the most successful results using Ti plasmid of the plant bacterium, Agrobacterium tumefaciens
130
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Examples
Plants/Crops
Organism that has acquired one or more genes From the same or another species.
Example : crops such as wheat, maize, fruits, (exported as food supply)
131
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Examples:
ANIMAL
Transgenic animal: transgenic salmon
Transgenic animals are animals that have been genetically engineered, a breeding approach that uses recombinant DNA techniques to create animals with new characteristics They are identified as a class of genetically modified organism (GMO)
136
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Genetically modified salmon is an Atlantic salmon, modified by adding a growth hormone regulating gene from a Pacific Chinook salmon and a promoter from an ocean pout to the Atlantic's 40,000 genes
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
ii. Production of Insulin The Vectors: E. coli
non pathogenic; harmless reproduce fast
can be engineered to produce large amount of insulin
•For medical purpose (treatment of diabetes) 139
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Definition Complementary DNA (cDNA)
A DNA molecule synthesised in vitro using an mRNA template catalyzed by an enzyme, reverse transcriptase. A cDNA molecule is identical to a native DNA, but lacks the non-coding regions, introns.
140
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Definition cDNA Library
A collection of cDNA molecules which represents only part of the genome. i.e. only the genes expressed in the cell used* “tissue-specific” – because it is constructed from mRNAs of one particular tissue
141
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Purposes Example…. mRNA of insulin – β-cells of islet of Langerhans
Growth hormones – pitutary gland of a kid
142
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Purposes Constructing cDNA library is easier compared to constructing a genomic library
Why?….
143
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Purposes Because…… cDNA is smaller compared to the original DNA and easy to manipulate
But…..
144
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Purposes
Disadvantage….. cDNA does not have the complete information on the genome of an organism.
But ….. Only has the genetic information for the genes that are expressed. 145
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Production of insulin Methods of Construction Overview
146
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Methods of Constructions
n
Isolation of mRNA from a particular cell to be used as a template for DNA synthesis
n
Addition of reverse transcriptase to synthesise a strand of cDNA molecule
147
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Methods of Constructions
n
Degradation of mRNA using ribonuclease or alkaline solution
n
Synthesis of the 2nd DNA strand catalysed by DNA polymerase
148
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Methods of Constructions
n
Ligation cDNA
n
Introduction of the rDNA
to linkers and then to a cloning vector
into the bacteria a cDNA library is completed 149
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Methods of Constructions
150
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Advantages Advantages of cDNA compared to genomic library i. does not contain non-coding introns ii. size of cDNA is smaller ~ easier to make a cDNA library iii. fewer types of clones are composed in cDNA library ~ screening process is simpler and faster iv. easier for researchers to study a particular gene responsible for a specialized function of a particular kind of cell
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Production of Insulin : The Procedures
DNA containing the insulin gene is isolated. DNA is spliced using restriction enzyme. To form DNA fragments With sticky ends Vector/plasmid from E. coli is isolated The vector is cut (same RE)/complementary ends Plasmid opens Plasmid and human gene fragment bear complementary sticky ends To produce linear plasmid with sticky ends 152
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Production of Insulin : The Procedures (continue…)
cDNA inserted into vector With aid of DNA ligase To form recombinant DNA/plasmid Recombinant DNA is transferred to host cell Transformation process Then screening is performed For cloning And amplification Insulin gene will be expressed Insulin is extracted from E.coli
153
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
iii. Environment
eg: degradation of oil spill
where the microorganisms esp. bacteria are used to convert the harmful toxic waste into the less harmful substance gene with controls the synthesis of Lipase is isolated from org. 70 genera of bacteria
useful genes of org. isolated and transferred into bacteria 154
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
• Genetically engineered Pseudomonas, an oil decomposing bacteria which can break down the main groups of hydrocarbon in oil.
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application Washington, March 30 : A new research has indicated that micro-organisms occurring naturally in coastal mudflats have an essential role to play in cleaning up pollution by breaking down petrochemical residues. The research, by Dr Efe Aganbi and colleagues from the University of Essex, reveals essential differences in the speed of degradation of the chemicals depending on whether or not oxygen is present. In aerobic conditions (where oxygen is present), benzene, toluene and naphthalene, which all occur in petroleum, were rapidly degraded by microbes. In the absence of oxygen, degradation was slower and only toluene was significantly broken down.
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
iii. Environment
eg: transgenic mosquitoes
GM mosquitoes were released into the environment in the Cayman Islands in 2009. GM mosquitoes in the eradication of Dengue fever was that the genetically modified mosquitoes were “engineered with an extra gene, or inserted bacterium, or have had a gene altered so that either their offspring are sterile and unable to spread dengue, or simply die
158
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
EXAMPLES OF GENE MANIPULATION
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
• Tomatoes were genetically engineered to be insect resistant
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
• Modified strawberries produce bacterial proteins that act as a natural antifreeze, providing protection from cold weather
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
• Modified potatoes produce edible vaccine to produce immunity against cholera.
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
•Transgenic – “Golden rice” produce yellow rice contain high concentrations of beta- carotene
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
Plants without pest resistance DNA of plant is combined with gene for pest resistance from other plant
Plants show pest resistance
Learning outcomes 8.3 Application of recombinant DNA Technology Briefly explain and give examples of recombinant DNA Technology application
