Cloning and Recombinant Dna
-
Upload
intanhardianti7 -
Category
Documents
-
view
28 -
download
2
description
Transcript of Cloning and Recombinant Dna
An Introduction to Cloning and An Introduction to Cloning and Recombinant DNARecombinant DNA
Prof. Arnaldo FerreiraProf. Arnaldo Ferreira
13.1 What Are Clones?
Clones • Genetically identical molecules, cells, or
organisms all derived from a single ancestor
Cloning• The production of identical copies of molecules,
cells, or organisms from a single ancestor
Cloning Higher Plants and Animals
Development of methods for cloning higher plants and animals represents a significant advance in genetic technology• Improving crops
• Producing domestic animals
Plants Can Be Cloned from Single Cells
1950s: Charles Steward grew individual carrot cells in the laboratory by using special nutrients
Single cells grew and divided to form a ball of undifferentiated cells (callus)
Calluses transferred to a different medium grew into full-size carrots (clones)
A Cloned Plant
Single cells grow and divide to form a callus
Animals Can Be Cloned by Several Methods
Embryo splitting• After in vitro fertilization, early embryonic cells are
divided and grown into clones
Nuclear transfer (cell fusion)• Enucleated eggs are fused with embryonic or
adult cells and grown into clones
• Dolly the sheep
Why is DNA Cloning Important?
DNA clones are used to find genes, map them, and transfer them between species
Cloning technology is used to find carriers of genetic disorders, perform gene therapy, and create disease-resistant plants
Keep In Mind
Cloned plants and animals are used in research, agriculture, and medicine
13.2 Cloning Genes Is a Multistep Process
Technology was developed to clone segments of DNA molecules, based on enzymes (restriction endonucleases) that recognize and cut DNA at specific nucleotide sequences
Recombinant DNA Technology
Recombinant DNA technology • Techniques in which DNA fragments are linked to
self-replicating vectors to create recombinant DNA molecules which are replicated in a host cell
What’s Needed to Clone DNA?
A way to cut DNA at specific sites
A carrier molecule to hold DNA for cloning
A place where the DNA can be copied (cloned)
DNA Can Be Cut at Specific SitesUsing Restriction Enzymes
Bacteria produce restriction enzymes to protect themselves from viral infections
Restriction enzymes • Bacterial enzymes that cut DNA at specific sites
Vectors are Carriers of DNA to be Cloned
Linking DNA segments produced by restriction-enzymes with vectors (plasmids or engineered viral chromosomes) produces recombinant DNA
Vectors • Self-replicating DNA molecules used to transfer
foreign DNA segments between host cells
Cloning Recombinant DNA
Recombinant DNA molecules are transferred into host cells; cloned copies are produced as the host cells grow and divide
Most common host cell: the bacterium E. coli
Cloned DNA molecules can be recovered from the host cells and purified for further use
E. coli
The recognition and cutting site for EcoRI
Plasmids
Plasmids used as vectors for cloning DNA
Steps in the Process of Cloning
DNA is cut with a restriction enzyme• Fragments produced end in specific sequences
Fragments are mixed with vector molecules cut by the same enzyme• DNA ligase joins recombinant DNA molecules
Steps in the Process of Cloning
Plasmid vectors with inserted DNA fragments are transferred into bacterial cells• Recombinant plasmids replicate and produce
many clones of the recombinant DNA molecule
Colonies carrying cloned recombinant DNA molecules are identified, collected, and grown• Host cells are broken open and recombinant
plasmids are extracted
Cloning Bacteria on Petri Plates
Each colony is a clone, descended from a single cell
Identifying Colonies With Recombinant DNA
Plasmid pBR322 has been engineered to carry two antibiotic-resistance genes with restriction sites, one for tetracycline, one for ampicillin
Colonies with human DNA inserted into the tetracycline gene will not grow on tetracycline plates, but will grow on ampicillin plates
Antibiotic-Resistance Genes on Plasmid pBR322
Two antibiotic-resistance genes, one for tetracycline, one for ampicillin
Keep In Mind
Cloning DNA uses techniques of biochemistry, genetics, and molecular biology
13.3 Cloned Libraries
A collection of cloned DNA sequences from one source is a library • Genomic library
• Chromosomal library
• Expressed sequence library
Libraries are resources for gene studies• Stored on yeast artificial chromosomes (YACs)
Storing a Genomic Library
Genomic library • A collection of clones that contains all the genetic
information in an individual
Yeast artificial chromosome (YAC) • Cloning vector with telomeres and a centromere
• Carries DNA fragments up to 1 million bases long
• Uses the eukaryote yeast as a host cell
Genetics in Society Asilomar: Scientists Get Involved
An international conference was held at Asilomar, California, to consider the possible dangers of recombinant DNA technology• In 1976, guidelines were set in place for
experiments using recombinant bacteria
• New guidelines were published in 1982
• No experiments are currently prohibited
13.4 Finding a Specific Clone in a Library
Clones for specific genes can be recovered from a library by using probes to screen the library
Probe • A labeled nucleic acid used to identify a
complementary region in a clone or genome
Keep In Mind
Finding a specific gene in a cloned library requires a molecular probe
13.5 A Revolution in Cloning: The Polymerase Chain Reaction
Polymerase chain reaction (PCR) • A method for amplifying DNA segments using
cycles of denaturation, annealing to primers, and DNA polymerase-directed DNA synthesis
PCR copies a DNA molecule without restriction enzymes, vectors, or host cells • Faster and easier than conventional cloning
First Step in PCR: Denaturation
1. DNA is heated to break the hydrogen bonds between the two polynucleotide strands• Two single-stranded DNA molecules serve as
templates
Second Step in PCR: Annealing
2. Short nucleotide sequences (primers for DNA replication) are mixed with the DNA and bind to complementary regions on single-stranded DNA • Takes place at lower temperature
• Primers are 20-30 nucleotides long, synthesized in the laboratory
Third Step in PCR: DNA Synthesis
3. The enzyme Taq polymerase is added to synthesize a complementary DNA strand• Taq is a DNA polymerase from a bacterium found
in hot springs
These three steps make up one PCR cycle
Many Uses for PCR
DNA to be amplified by PCR does not have to be purified and can be present in small amounts• Used in clinical diagnosis, forensics, conservation
• Samples can be small or old (insects in amber)
Keep In Mind
The polymerase chain reaction (PCR) copies DNA without cloning
13.6 Analyzing Cloned Sequences
Cloned sequences are characterized in several ways, including Southern blotting and DNA sequencing
Southern blot • A method for transferring DNA fragments from a
gel to a membrane filter, developed by Edwin Southern for use in hybridization experiments
Southern Blotting Can Be Used to Analyze Cloned Sequences
Southern blotting uses gel electrophoresis to separate DNA fragments• DNA fragments migrate through the gel from a
negative pole to a positive pole
• Small fragments move faster than large fragments
Radioactive probes identify blotted bands
DNA Sequencing Can Be Done for an Entire Genome
DNA sequencing • A technique for determining the nucleotide
sequence of a fragment of DNA
• Basic method used in genome projects
There are several ways to sequence DNA
Automated Sanger Method
DNA is separated into strands
DNA polymerase, a primer, and four kinds of altered nucleotides are added• Each nucleotide fluoresces a different color
When an altered nucleotide is added, synthesis stops; strands of every length accumulate• Fragments are separated by length and scanned
with a laser that reveals the fluorescent tag
Genetic Journeys: DNA Sequencing
In 1977, Fred Sanger sequenced the 5,400 nucleotides in the genome of a virus
Automated methods allowed the human genome (3.2 billion nucleotides) to be sequenced
DNA sequencing is one of the basic methods in recombinant DNA technology