Biology Students Resources SB015 1 | KMPk CHAPTER 8: RECOMBINANT DNA TECHNOLOGY SUBTOPIC : 8.1 Recombinant DNA Technology LEARNING OUTCOMES : a) Define recombinant DNA technology. b) Define and explain the tools used in recombinant DNA technology, target DNA, restriction enzymes, DNA cloning vector, host cell and modifying enzymes. (c) Explain restriction enzyme and examples of enzymes that produce sticky ends. (EcoRI: G AATTC) and blunt ends (SmaI : CCC GGG) (d) Explain the characteristics of plasmid as cloning and expression vector (e) Explain the characteristic of E.coli as host cell and its characteristics (f) Explain modifying enzyme and its function; (i) DNA ligase for DNA ligation (ii) Taq polymerase for DNA amplification using PCR. . MAIN IDEAS /KEY POINT EXPLANATION NOTES Recombinant DNA technology It’s a technology that forms a new combination of DNA when segments of DNA from two different sources (often different species) in vitro. Purpose • Enable scientists to obtain copies of specific DNA segment for the purpose of studying it. • Modifying the DNA of an organism to produces new genes with new traits. Tools Used in Cloning 1. Target DNA (Genes of interests). It’s a fragment of chromosomal to be cloned. An enzyme must be used to cleave fragments that containing genes of interests. 2. Restriction Enzymes / Restriction of endonucleases. This enzyme is extracted from a specific bacteria, for example E.coli. Naturally it is used to cut/splice viral DNA into small fragments at specific base sequence/ restriction sites. Most of the base are palindromic. Palindromic is a base sequence of one strand reads the same as its complement strand in opposite direction. BIOLOGY SCORE
13
Embed
CHAPTER 8: RECOMBINANT DNA TECHNOLOGY · EcoRI 5’-GAATTC-3’ 3’-CTTAAG-5’ BamHI 5’-GGATCC-3’ 3’-CCTAGG-5’ SmaI 5’-GGGCCC-3’ 3’-CCCGGG-5’ The list and explanation
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Biology Students Resources SB015
1 | KMPk
CHAPTER 8: RECOMBINANT DNA TECHNOLOGY
SUBTOPIC : 8.1 Recombinant DNA Technology
LEARNING OUTCOMES : a) Define recombinant DNA technology. b) Define and explain the tools used in recombinant DNA technology, target
DNA, restriction enzymes, DNA cloning vector, host cell and modifying
enzymes.
(c) Explain restriction enzyme and examples of enzymes that produce sticky ends.
(EcoRI: G AATTC) and blunt ends (SmaI : CCC GGG) (d) Explain the characteristics of plasmid as cloning and expression vector
(e) Explain the characteristic of E.coli as host cell and its characteristics
(f) Explain modifying enzyme and its function; (i) DNA ligase for DNA ligation (ii) Taq polymerase for DNA
amplification using PCR.
.
MAIN IDEAS /KEY POINT
EXPLANATION NOTES
Recombinant
DNA technology
It’s a technology that forms a new combination of DNA when
segments of DNA from two different sources (often different
species) in vitro.
Purpose
• Enable scientists to obtain copies of specific DNA segment
for the purpose of studying it.
• Modifying the DNA of an organism to produces new genes
with new traits.
Tools Used in Cloning
1. Target DNA (Genes of interests).
It’s a fragment of chromosomal to be cloned. An enzyme
must be used to cleave fragments that containing genes of
interests.
2. Restriction Enzymes / Restriction of endonucleases.
This enzyme is extracted from a specific bacteria, for example
E.coli. Naturally it is used to cut/splice viral DNA into small
fragments at specific base sequence/ restriction sites. Most of
the base are palindromic. Palindromic is a base sequence of one
strand reads the same as its complement strand in opposite
direction.
BIOLOGY
SCORE
Biology Students Resources SB015
2 | KMPk
3. DNA cloning vector
A small pieces of DNA which a foreign DNA fragment can
be inserted. For examples, plasmid, bacteriophage, cosmid
and YACs. Every vector must have these characteristics:
1. Able to accept foreign DNA in multiple cloning sites
(MCS)
2. Able to replicate freely in host cell.
Present of origin of replication initiation -ori gene
3. Possess selectable genetic marker
a. resistance to antibiotic.
eg: ampR, tetR, kanR
b. lacZ gene
encode for B- galactosidase
4. Host Cell
A host cell is a cell that has been introduced
with DNA (or RNA), such as a bacterial cell acting as a host
cell for the DNA isolated from a bacteriophage. This cell is
utilized from the DNA cloning to accept, maintain and allow
the replication of cloning vector. This cell needs to have
these characteristics:
1. Able to receive DNA through the transformation
processes.
2. Able to maintain the structure of DNA recombinant
SUBTOPIC : 8.2 Methods in Gene Cloning LEARNING OUTCOMES : a) Overview using diagram to show the steps in gene cloning by using plasmid b) Describe the steps in gene cloning by using plasmid as the vector
(i) isolation of gene
(ii) cleave/cut
(iii) insertion (iv) transformation and amplification
(v) screening (blue/white screening)
MAIN IDEAS /KEY POINT
EXPLANATION NOTES
Cloning
Cloning is the process of producing genetically identical individuals
of an organism either naturally or artificially.
Gene Cloning
Is a process that produce many copies of a gene of interest by
making many identical copies of a gene by inserting the gene into a
living host cell (bacteria). These copies can be used in sequencing
the gene.
• 2 methods to copies the DNA fragment:
a. Cloning
b. Polymerase Chain Reaction (PCR)
Steps in Gene
Cloning (Refer
to Campbell,
2011, (9th Ed.),
Pg. 445)
1. Isolation of gene and vector (plasmid) from source (different
organisms).
• Isolation of plasmid (as a vector) from bacteria.
BIOLOGY
SCORE
Biology Students Resources SB015
7 | KMPk
• Isolation of target DNA or gene of interest from sources
Eg: Human cells, Plant cells, Animal cells
2. Cleave or cut of DNA/gene of interest and plasmid by using the
same restriction enzyme
• The target DNA and plasmid are cut at palindromic
sequence (restriction site) with the same restriction enzyme.
Single cut within the lacZ gene, caused the plasmid to open
and the disruption of the gene. However, many cuts within
the target DNA produced thousands of fragments with
different sizes.
3. Insertion of gene of interest into plasmid (vector).
• Mix the cut plasmid and DNA fragments allowing base pair
between their complementary sticky ends by forming
hydrogen bonds. DNA ligase is added to seal them together
at the sugar phosphate backbones of the fragments whose
sticky ends have base-paired with phosphodiester bond.
Thus, forming a recombinant DNA.
Biology Students Resources SB015
8 | KMPk
• BUT, not all plasmid will join with the target DNA. This
caused two types of recombinant DNA:
Non recombinant plasmid
Recombinant plasmid
4. Transformation of recombinant DNA into host cells (bacteria)
and amplification.
– Host cells take up recombinant DNA after being
introduced to it. BUT, not all host cells will take in
the recombinant DNA. Only a small proportion of
bacteria will be transformed.
• There will be THREE conditions:
1. Transformed with non-recombinant
plasmid
2. Transformed with recombinant
plasmid
3. Not transformed
Then, they are mixed in a medium containing calcium chloride. This
solution caused the bacterial cell wall to become permeable. The
host cells reproduce by binary fission and amplify the target DNA.
Inserted gene of
interest
Biology Students Resources SB015
9 | KMPk
5. Screening
The mixed bacteria are plate on nutrient-containing agar
medium supplemented with ampicillin and X-gal, a molecule
resembling lactose.
– Purpose: To identify bacterial colonies that carry
gene of interest / target DNA = Blue-white
screening.
• A plasmid vector contains two genes encoding for :
– Resistance to antibiotic (ampR)
– Enzyme β-galactosidase (lacZ)
–
• When cultured in medium containing ampicilin and X-gal:
Bacteria without plasmid fail to grow because of
none ampR gene – not resistance to antibiotic
• Bacteria with plasmid live but with TWO conditions:
– Contain non-recombinant plasmids.
• lacZ gene that encodes for β-galactosidase is
functioning
• Hydrolyzed X-gal BLUE colonies
– Contain recombinant plasmids.
• lacZ gene that encodes for β-galactosidase is
disrupted.
• X-gal not hydrolyzed WHITE colonies.
Biology Students Resources SB015
10 | KMPk
Biology Students Resources SB015
11 | KMPk
BIOLOGY
SCORE
CHAPTER 8 : RECOMBINANT DNA TECHNOLOGY
SUBTOPIC : 8.3 Application of Recombinant DNA Technology LEARNING OUTCOMES a) Briefly explain applications of Recombinant DNA Technology in mass
production of insulin using cDNA. b) Describe the steps in production of insulin using cDNA.
MAIN IDEAS
/KEY POINT EXPLANATION NOTES
Application Of
Recombinant
DNA
Technology
• Many fields benefit from DNA technology and genetic engineering.
- Agriculture
- Forensic
- Medicine – insulin for diabetics
- Environment
Biology Students Resources SB015
12 | KMPk
Complementary
DNA (cDNA)
cDNA is DNA molecule made in vitro using mRNA as a template
and the enzyme reverse transcriptase.
A cDNA molecule lack introns compared to DNA of the genome
Can be used directly to express the proteins.
cDNA is often used to clone eukaryotic genes in prokaryotes
cDNA is used because:
cDNA represents the expression of the genes without the
introns.
there is no process for RNA splicing in bacteria cell.
Production of
insulin
1. mRNA for insulin is isolated from the beta cells of islets of
Langerhans in the pancreas.
2. Reverse transcriptase enzyme is added to synthesis a cDNA by
using mRNA as template. mRNA strand then discarded by using
mRNA-degrading enzyme.
3. DNA polymerase enzymes is added and synthesizes a second
DNA strand, complementary to cDNA in vitro.
• cDNA : A DNA molecule made in vitro using mRNAas a template and the enzyme reverse transcriptase.
• A cDNA molecule therefore corresponds to a gene, but lacks the introns present in the DNA of the genome.
• Can be used directly to express the proteins
encoded by them. 47
Complementary DNA ( cDNA )
Biology Students Resources SB015
13 | KMPk
4. Restriction enzyme ( BamHI ) is used to cut the cDNA and isolate
just the sequence that encodes for the insulin protein.
5. Plasmid removed from a bacterial cell is cut at a specific site using
the same restriction enzyme (BamHI)
6. The cDNA is inserted to the plasmid using DNA ligase. The
recombinant plasmid carrying the human DNA for insulin.
7. The recombinant DNA then transform into the host(E. coli) by
transformation.
8. The bacterium E. coli with its recombinant plasmids, is allowed
to reproduce and undergo screening process.
9. Finally, a lot of bacterial clone carrying many copies of the gene