Introduction to Cloning and Recombinant DNA Technology David Bedwell, Ph.D. Department of Microbiology Office telephone: 934-6593 Email: [email protected] The Powerpoint slides for this lecture are available for download at: http://www.microbio.uab.edu/bedwell/index4.html
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David Bedwell, Ph.D.
Department of Microbiology
Office telephone: 934-6593
Email: [email protected]
The Powerpoint slides for
this lecture are available for download at:
Reference: Molecular Biology of the Cell, 5th Edition, by Alberts et al., published by Garland Science, 2008.
Plasmid OutlineDNA is the genetic material of most organisms (from bacteria to humans)
Chromosome: Most bacteria have one circular DNA chromosome ranging in size from 1,000 to 8,000 kilobase pairs.
Plasmid: Extrachromosomal genetic element also made of a circular DNA molecule.
Bacterial Genome: The collection of all of the genes present on the bacteria’s chromosome or its extrachromosomal genetic elements.
Only in RNA,
Clone: a collection of molecules or cells, all identical to an original molecule or cell
Examples of Palindromes:
Dogma: I am God
Never odd or even
Too bad – I hid a boot
Rats live on no evil star
No trace; not one carton
Was it Eliot's toilet I saw?
Murder for a jar of red rum
Some men interpret nine memos
Campus Motto: Bottoms up, Mac
Go deliver a dare, vile dog!
Madam, in Eden I'm Adam
Oozy rat in a sanitary zoo
Ah, Satan sees Natasha
Lisa Bonet ate no basil
Do geese see God?
God saw I was dog
Cuts usually occurs at
a palindromic sequence
SmaI: produces blunt ends
5´ CCCGGG 3´
3´ GGGCCC 5´
EcoRI: produces sticky ends
5´ GAATTC 3´
3´ CTTAAG 5´
Why the funny names?
5’ --AAGCTT-- 3’ 5’ --A AGCTT--3’
3’ --TTCGAA-- 5’ 3’ –TTCGA A--5’
5’--GGTACC-- 3’ 5’ –GGTAC C-- 3’
3’--CCATGG-- 5’ 3’ –C CATGG-- 5’
5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’
3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’
5’ --A AGCTT--3’ 5’ --AAGCTT-- 3’
3’ --TTCGA A--5’ 3’ --TTCGAA-- 5’
DNA fragments with blunt ends generated by different enzymes can be ligated together (with lower efficiency), but usually cannot be re-cut by either original restriction enzyme.
No longer palindromic, so not cut by BamHI or BglII
AmprPlasmids – vehicles for cloning
Older cloning vector
Newer cloning vector
DNA synthesis requires a 3´-OH to make the next phosphodiester bond during DNA synthesis
ddNTPs block formation of the next phosphodiester bond during DNA synthesis
A 3´-OH on the last ribose is needed for DNA synthesis
A nucleotide-specific stop in DNA synthesis
Fluorescent dye coupled to reaction allows visualization of di-deoxy termination events by means of a laser that detects the colored product.
This shows four different reactions as done with the old manual sequencing.
3.2x109 nucleotide pairs
Took just 4 months,
$1.5 million to obtain the entire DNA sequence of James Watson.
The genomes of many organisms have been sequenced each reaction
-Purify genomic DNA
-Digest with restriction enzyme
-Run agarose gel
Consider two alleles of a gene. Allele A has 3 BamHI sites, while allele a has only two BamHI sites.
1 2 3
70% of carriers of the sickle cell gene have a 13.0 kb HpaI fragment.
30% of carriers have 7.0 kb HpaI fragment
A specific hemoglobin mutation
Pro Glu Pro Val
CCT GAG CCT GTG
DdeI site no DdeI site
[DdeI cuts at CTNAG]
Gene encoding sickle cell b-subunit
Gene encoding Wild type b-subunit
Polymerase Chain Reaction (PCR) Outline
A thermophilic (heat-loving) bacteria called Thermus aquaticus is the source of Taq DNA polymerase used in PCR reactions.
A typical PCR protocol Outline
5´PCR cycle 28 – ~1 billion strands
2 original strands.
28 strands starting with primer A, but with no end.
28 strands starting with primer B, but with no end.
~500,000,000 strands starting with primers A (5´) and ending with primer B (referred to as unit-length strand in previous figure).
~500,000,000 strands starting with primer B (5´) and ending with primer A (referred to as unit-length strand in previous figure).
-dot blot (spot hybridization) with oligonucleotides specific for known mutation.
-restriction enzyme analysis (RFLP).
-direct sequencing of DNA.
-Tay-Sachs disease, phenylketonurea, cystic fibrosis, hemophilia, Huntingdon's disease, Duchenne muscular dystrophy (DMD).
VNTR is based on hypervariable microsatellite sequence polymorphisms within the human genome. These sequences (e.g., CACACA …) are found in many locations in the human genome and vary greatly from person to person.
Individuals A & C are excluded by this analysis. The samples from individual B will be subjected to further tests.