Chapter 20: Recombinant DNA Technology, Lecture Notes Flashcards
Modern techniques
Developed in 70’s
Virtual revolution in biotechnology
First commercial approval was 1982 for bacterial produced human insulin
Has led to an explosion of this industry
Clone definition
Identical organisms
Identical cells
Identical molecules
Descended from a single ancestor
Cloning
Produces many identical copies
Use for research
Use commercially
Recombinant DNA….
What is it?
Combination of DNA molecules not naturally found together
Methods derived from nucleic acid biochemistry
recombinant DNA technology
A collection of methods used to create DNA molecules by in vitro ligation of DNA from two different organisms, and the replication and recovery of such recombinant DNA molecules.
Recombinant DNA method
Purify target DNA
Digest target DNA with enzymes (restriction endonucleases) that cut DNA at specific sequences
Join these fragments with other carrier DNA molecules (vectors)
Results in RECOMBINANT DNA molecule.
Recombinant DNA molecule is transferred into a host cell for replication.
Recombinant DNA passed on within population of host cells, making more copies.
Cloned DNA recovered, analyzed etc.
Cloned DNA within host cell can be transcribed, mRNA translated, gene product isolated (eg. insulin).
It’s hard to overestimate how this methodology has changed biological research
restriction endonuclease
A bacterial nuclease that recognizes specific nucleotide sequences in a DNA molecule, often a palindrome, and cleaves or nicks the DNA at those sites.
Provides bacteria with a defense against invading viral DNA.
Restriction endonuclease are widely used in the construction of recombinant DNA molecules.
palindrome
In genetics, a sequence of DNA base pairs that reads the same backward or forward. Because strands run antiparallel to one another in DNA, the base sequences on the two strands read the same backward and forward when read from the 5’ end. For example: 5’-GAATC-3’ 3’-CTTAAG-5’ .
Palindromic sequences are noteworthy as recognition and cleavage sites for restriction endonucleases.
Recombination:
First Steps
DNA isolated from cells
Many methods are used to isolate nucleic acids
Fractionation methods separate nucleic acids from proteins and other cellular debris
Many simple commercial kits are (Qiagen) available for this today to extract DNA from countless sources
DNA extracted with DNeasy tissue extraction kit (Qiagen Inc.)
Lanes 2, 4, 6, 8, 10, 12, 14: Undigested genomic DNA, green arrows
Lanes 1 & 15: DNA ladder, red arrows
Specific DNA Digestion
The restriction enzyme EcoRI recognizes and binds to the palindromic nucleotide sequence GAATTC.
Cleavage of the DNA at this site produces complementary single-stranded tails.
These single-stranded tails anneal with single-stranded tails from other DNA fragments to form recombinant DNA molecules.
Restriction Endonucleases (RE)
Each RE recognizes a specific sequence of DNA (recognition site), which is a palindrome (same sequence 5’->3’ on opposite strands)
reannealing
Formation of double-stranded DNA molecules from denatured single strands.
RE specificity, source, and naming
Some common restriction enzymes, with their recognition sequences, cutting sites, cleavage patterns, and sources.
Popular REs include EcoRi, Hindlll, and BamHI.
All of these RE create “sticky” ends.
All of these recognize 6 base sites: “6-cutters”
RE recognizing 4 base sites:
“4-cutters”
Some, eg. TaqI, create “sticky” ends.
Some, eg. AluI & HaeIII, create “blunt” ends.
Annealing allows recombinant DNA molecules to form by complementary base pairing.
The 2 strands are not covalently bonded until DNA ligase comes along to fill the gaps.
DNA from different sources is cleaved with EcoRI and mixed to allow annealing to form recombinant molecules. The enzyme DNA ligase then chemically bonds these annealed fragments into an intact recombinant DNA molecule.
Fragment Ligation
Join the ends of DNA together
Complementary (“sticky”) ends will anneal with one another
2 different molecules cut with the same RE can anneal to one another
DNA ligase forms covalent links to form recombinant molecule
Vectors
Used to carry DNA fragments
Used as a way to get genetic material in (a vector) to a host cell where the vector & fragment can be replicated
Vector requirements
Ability to replicate itself and the DNA fragment it carries
RE sites in vector
Selectable marker (eg. antibiotic resistance) to distinguish hosts with and without vector
Easy to recover vector from host cells
The plasmid pUC18 offers several advantages as a vector for cloning.
Because of its small size, it accepts relatively large DNA fragments for cloning.
It replicates to a high copy number, and has a large number of restriction sites in the polylinker, located within a lacZ gene.
Bacteria carrying pUC18 produce blue colonies when grown on media containing Xgal.
DNA inserted into the polylinker site disrupts the lacZ gene; this results in white colonies and allows direct identification of colonies carrying cloned DNA inserts.
First generation vectors
pUC vectors (5-10kb)
Derived from bacterial plasmids
Replicates 500X in host
Large number of RE sites (polylinker)
Selection system (ampicillin gene)
Insert discrimination system (lacZ gene)
b-galactosidase
A bacterial enzyme, encoded by the lacZ gene, that converts lactose into galactose and glucose.
LacZ insert identification
LacZ gene is part of bacterial Lac operon
Produces Beta-galatosidase that metabolizes lactose to produce glucose and galactose
Beta-galactosidase action results in a blue by-product when X-gal is present in the growth medium (color indicator)
LacZ insert identification method
Grow bacteria with X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside)
When intact, LacZ gene product results in blue colonies
However, if a DNA fragment is inserted in polylinker region, LacZ gene is inactivated
Result: no X-gal metabolism ==>WHITE COLONIES
Plasmid Cloining Overview
Plasmid vectors are isolated and cut with a restriction enzyme.
The DNA to be cloned is cut with the same restriction enzyme, producing a collection of fragments.
These fragments are spliced into the vector and transferred to a bacterial host for replication.
Bacterial cells carrying plasmids with DNA inserts are identified by growth on selective medium and isolated.
The cloned DNA is then recovered from the bacterial host for further analysis.
Next vector system
Need to handle larger insert (10-15kb)
Bacterial virus (phage) vectors
Eg., M13, Lambda phage vectors
Lambda phage vector system
Central third of phage DNA expendable
Cut out central third of lambda
Leaves left arm, right arm, central region
Isolate arms, recombine with DNA fragment of interest, and ligate together
Introduce packaged ligated phage to host cell
Phage replicates in host, makes phage particles that contain inserts
Form plaques on bacterial lawns
Can isolate & use for further analysis
Phage lambda as a vector.
DNA is extracted from a preparation of lambda phage, and the central gene cluster is removed by treatment with a restriction enzyme.
The DNA to be cloned is cut with the same enzyme and ligated into the arms of the lambda chromosome.
The recombinant chromosome is then packaged into phage proteins to form a recombinant virus.
This virus infects bacterial cells and replicates its chromosome, including the DNA insert.
Cosmids
Can carry up to 50kb inserts
Cosmid: part phage, part plasmid
Engineered vector
Ligate fragments into cosmid, package into protein heads, infect hosts
The cosmid pJB8 contains a:
bacterial origin of replication (ori),
a single cos site (cos),
an ampicillin resistance gene (amp, for selection of colonies that have taken up the cosmid),
and a region containing 4 restriction sites for cloning (BamHI, EcoRI, ClaI, and HindIII).
Because the vector is small (5.4 kb long), it can accept foreign DNA segments between 33 and 46 kb in length.
The cos site allows cosmids carrying large inserts to be packaged into lambda viral coat proteins as though they were viral chromosomes.
The viral coats carrying the cosmid can be used to infect a suitable bacterial host, and the vector, carrying a DNA insert, will be transferred into the host cell. Once inside, the ori sequence allows the cosmid to replicate as a bacterial plasmid.
Bacterial artificial chromosomes (BACs)
Good for large fragments
Based on F(ertility) factor of bacteria
Clone up to 300kb
Multiple cloning sites, selective markers, promoters to express inserted genes
A bacterial artificial chromosome (BAC) caption
The polylinker carries a number of unique restriction sites for the insertion of foreign DNA.
The arrows labeled T7 and Sp6 are promoter regions that allow expression of genes cloned between these regions.
Yeast artificial chromosomes (YACs)
Have telomeres, ori, and centromere
Joined to selectable markers, cluster of RE sites
Clone inserts 100kb to 1Mb
Important vector for genomic sequencing, eg. Human Genome Project
YAC
A cloning vector in the form of a yeast artificial chromosome, constructed using chromosomal components including telomeres (from a ciliate), and centromeres, origin of replication,and marker genes from yeast. YACs are used to clone long stretches of eukaryotic DNA.
Cloning into a yeast artificial chromosome
The synthetic chromosome contains telomere sequences (TEL), a centromere (CEN4) derived from yeast chromosome 4, and an origin of replication (ori).
These elements give the cloning vector the properties of a chromosome. TRP1, SUP4, and URA3 are yeast genes that are selectable markers for the left and right arms of the chromosome, respectively.
Within the SUP4 gene is a restriction site for the enzyme SnaB1.
Two BamH1 sites flank a spacer segment.
Cleavage with SnaB1 and BamH1 breaks the artificial chromosome into two arms.
The DNA to be cloned is treated with the same enzyme, producing a collection of fragments.
The arms and fragments are ligated together, and the artificial chromosome inserted into yeast host cells.
Because yeast chromosomes are large, the artificial chromosome accepts inserts in the million base pair range (Mb 5 megabase).
A Ti plasmid designed for cloning in plants
Segments of Ti DNA, including those necessary for opine synthesis and integration, are combined with bacterial segments that incorporate cloning sites and antibiotic resistance genes (kanR and tetR).
The vector also contains an origin of replication (ori) and a lambda cos site that permits recovery of cloned inserts from the host plant cell.
Ti plasmid
A bacterial plasmid used as a vector to transfer foreign DNA to plant cells.
Gene Transfer in Eukaryotic Cells:
Plant cell hosts
Done using bacterial plasmids as vectors
Gene Transfer in Eukaryotic Cells:
Mammalian cell hosts
Encapsulation or endocytosis to transfer DNA to mammalian cell hosts
Micromanipulation
Cloning in Yeast cells
Eukaryotic, acts like bacteria for growth and manipulation
Extensively studied, entire genome seq.
To study function of eukaryotic proteins, need a eukaryotic system
Yeast considered safe
Transgenic definition
Plants or animals carrying a foreign gene are transgenic
transgenic organism
An organism whose genome has been modified by the introduction of external DNA sequences into the germ line.
Cell Free Cloning:
PCR
Polymerase Chain Reaction
Cloning without need for host cell
Making many copies (millions)
polymerase chain reaction (PCR)
A method for amplifying DNA segments that depends on repeated cycles of denaturation, primer annealing, and DNA polymerase–directed DNA synthesis.
Kary Mullis
Developed PCR method
Parts known previously
Hard to overestimate the effect of PCR on how molecular genetics is done today
1993 Nobel Prize
Basic PCR Procedure
Denature target DNA
Anneal specific primers to flank DNA target of interest
Extend primers using Taq DNA polymerase
Repeat 25-40X
25 cycles results in 10^6 increase in target
Basic PCR Procedure:
Taq DNA polymerase
Taq polymerase is key to PCR, derived from thermophilic bacteria Thermus aquaticus
A hot spring bacteria, the polymerase works at elevated temps: 68-72C
Taq polymerase survives high temperatures (eg. 95C)
PCR amplification
In the polymerase chain reaction, the target DNA is denatured into single strands; each strand is then annealed to a short, complementary primer.
The primers are synthetic oligonucleotides that are complementary to sequences flanking the region to be amplified.
DNA polymerase and nucleotides extend the primers in the 3’ direction, using the single-stranded DNA as a template.
The result is a double-stranded DNA molecule with the primers incorporated into the newly synthesized strand.
In a second PCR cycle, the products of the first cycle are denatured into single strands, primers are annealed, and DNA polymerase then synthesizes new strands.
Repeated cycles can amplify the original DNA sequence by more than a millionfold.
PCR Advantages
FAST, no cell based cloning required
Done by programmable machine, essentially a glorified heating/cooling block
PCR is VERY sensitive, can work with only a few targets
Great for forensics, degraded DNA, etc
PCR Limitations
PCR is VERY sensitive, can lead to false positives due to contaminating DNA
Standard PCR requires knowledge of DNA sequences surrounding region of interest
Other uses of PCR
Identification of RE variants
Microsatellite analysis
Screening for genetic disorders
Diagnostic screening for infectious organisms
Forensics
Paleobiology
