an introduction to ant dna technology
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An introduction to
recombinant DNAtechnology
S.K.Kashyap
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contents
1. rDNA technology makes it possible to clonegenes for basic research and commercialapplications: an overview
2. Restriction enzymes are used to makerecombinant DNA
3. Genes can be cloned in recombinant DNA vectors:a closer look
4. Cloned genes are stored in DNA libraries
5. The polymerase chain reaction (PCR) cloned DNAdirectly in vitro
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The mapping and sequencing of the human genome hasbeen made possible by advances in DNA technology.
Progress began with the development of techniques formaking recombinant DNA, in which genes from two
different sources - often different species - arecombined in vitro into the same molecule.
These methods form part ofgenetic engineering, thedirect manipulation of genes for practical purposes.
Applications include the introduction of a desired gene intothe DNA of a host that will produce the desired protein.
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DNA technology has launched a revolution in biotechnology, themanipulation of organisms or their components to make usefulproducts.
Practices that go back centuries, such as the use of microbes to makewine and cheese and the selective breeding of livestock, are examplesof biotechnology.
Biotechnology based on the manipulation of DNA in vitro differs fromearlier practices by enabling scientists to modify specific genes andmove them between organisms as distinct as bacteria, plants, andanimals.
DNA technology is now applied in areas ranging from agricultureto criminal law, but its most important achievements are in basic
research.
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Cloning - a definition
From the Greek - klon, a twig
An aggregate of the asexually produced progenyof an individual;a group of replicas of all or part
of a macromolecule (such as DNA or anantibody)
An individual grown from a single somatic cell ofits parent & genetically identical to it
Clone: a collection of molecules or cells,all identical to an original molecule or cell
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DNA CLONING
A method for identifying and purifying a
particular DNA fragment (clone) of interest
from a complex mixture of DNA fragments,
and then producing large numbers of the
fragment (clone) of interest.
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Gene cloning
When DNA isextracted from an
organism, all itsgenes are obtained
In gene (DNA)cloning a particular
gene is copied(cloned)
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Why Clone DNA?
A particular gene can be isolated and itsnucleotide sequence determined
Control sequences of DNA can be identified &analyzed
Protein/enzyme/RNA function can beinvestigated
Mutations can be identified, e.g. gene defectsrelated to specific diseases
Organisms can be engineered for specificpurposes, e.g. insulin production, insect
resistance, etc.
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Sources of DNA for
Cloning
1) Chromosomal DNA
2) RNA converted to cDNA
3) chemically synthesized genes
4) PCR-amplified DNA
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In order to have enough DNA to work with for a single gene or sequence,you must have a way to clone, or reproduce many exact copies of that
gene.This is called molecular cloningThe DNA containing the gene must be isolated away from its genomiccontextThat should be simple but there are big problems
Molecular Cloning
Gene ofinterest
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To study a particular gene, scientistsneeded to develop methods to isolate onlythe small, well-defined, portion of a
chromosome containing the gene.
Techniques for gene cloning enablescientists to prepare multiple identical
copies of gene-sized pieces of DNA.
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An overview
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The potential uses of cloned genes fall into two generalcategories.
First, the goal may be to produce a protein product.
For example, bacteria carrying the gene for human growthhormone can produce large quantities of the hormone for
treating stunted growth.
Alternatively, the goal may be to prepare many copies ofthe gene itself.
This may enable scientists to determine the genes nucleotidesequence or provide an organism with a new metaboliccapability by transferring a gene from another organism.
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Gene cloning and genetic engineering were made possibleby the discovery ofrestriction enzymes that cut DNAmolecules at specific locations.
In nature, bacteria use restriction enzymes to cut foreignDNA, such as from phages or other bacteria.
Most restrictions enzymes are very specific, recognizingshort DNA nucleotide sequences and cutting at specificpoint in these sequences.
Bacteria protect their own DNA by methylation.
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Restriction endonucleases
Also called restriction enzymes
Occur naturally in bacteria
Hundreds are purified and available commercially
Named for bacterial genus, species, strain, and type
Example: EcoRI
Genus: Escherichia
Species: coli
Strain: R
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Recognize specific base sequences in DNA
Cut DNA at those recognition sites
Restriction endonucleases
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Enzymes recognize specific 4-8 bp sequences
EcoRI 5GAATTC3
3CTTAAG5
Recognition sites have symmetry Some enzymes cut in a staggered fashion Some enzymes cut in a direct fashion
PvuII 5CAGCTG3
3GTCGAC5
Restriction Enzyme Recognition Site
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Products generated by restriction enzymes
COHESIVE END CUTTERS (staggered cuts):
Enzyme Recognition Site Ends of DNA After
Cut
EcoRI 5GAATTC3 5G AATTC3
3CTTAAG5 3CTTAA G5
PstI 5CTGCAG3 5CTGCA G3
3GACGTC5 3G ACGTC5
BLUNT END CUTTERS (direct cuts):
Enzyme Recognition Site Ends of DNA After Cut
HaeIII 5GGCC3 5GG CC3
3CCGG5 3CC GG5
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Average distance between cuts is:
4
n
where n is number of bps in recognition site.
4-base cutter: 44 = 256 bp
5-base cutter: 45 = 1,024 bp 6-base cutter: 46 = 4,096 bp 8-base cutter: 48 = 65,536 bp
Frequency of cutting
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Each restriction enzyme cleaves a specificsequences of bases or restriction site.
These are often a symmetrical series of four to eightbases on both strands running in opposite directions.
If the restriction site on one strand is 3-CTTAGG-
5,
the complementary strand is 5-GAATTC-3.
Because the target sequence usually occurs (by
chance) many times on a long DNA molecule, anenzyme will make many cuts.
Copies of a DNA molecule will always yield the same setof restriction fragments when exposed to a specific
enzyme.
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Restriction enzymes cut covalent phosphodiesterbonds of both strands, often in a staggered waycreating single-stranded ends sticky ends.
These extensions will form hydrogen-bonded base pairswith complementary single-stranded stretches on otherDNA molecules cut with the same restriction enzyme.
These DNA fusions can be made permanent byDNA ligase which seals the strand by catalyzingthe formation of phosphodiester bonds.
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Recombinant plasmids are produced by splicingrestriction fragments from foreign DNA intoplasmids.
These can be returned relatively easily to bacteria.
The original plasmid used to produce recombinant DNA iscalled a cloning vector, which is a DNA molecule that can
carry foreign DNA into a cell and replicate there.
Then, as a bacterium carrying a recombinantplasmid reproduces, the plasmid replicates within it.
3. Genes can be cloned in DNA vectors: a closer look
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Bacteria are most commonly used as host cells forgene cloning because DNA can be easily isolated and
reintroduced into their cells.
Bacterial cultures also grow quickly, rapidly
replicating the foreign genes.
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The process ofcloning a humangene in a bacterialplasmid can bedivided into five
steps.
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1. Isolation of a vector and gene-sourceDNA.
The source DNA comes from human tissue cells.
The source of the plasmid is typically E. coli.
This plasmid carries two useful genes:
ampR, conferring resistance to the antibiotic ampicillin
lacZ, encoding the enzyme beta-galactosidase whichcatalyzes the hydrolysis of sugar.
The plasmid has a single recognition sequence, within thelacZgene, for the restriction enzyme used.
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2. Insertion of DNA into the vector.
By digesting both the plasmid and human DNA with the samerestriction enzyme we can create thousands of human DNAfragments, one fragment with the gene that we want, and withcompatible sticky ends on bacterial plasmids.
After mixing, the human fragments and cut plasmids formcomplementary pairs that are then joined by DNA ligase.
This creates a mixture of recombinant DNA molecules.
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3. Introduction of the cloningvector into cells.
Bacterial cells take up the recombinant
plasmids by transformation. These bacteria are lacZ-, unable to hydrolyze
lactose.
This creates a diverse pool of bacteria,some bacteria that have taken up thedesired recombinant plasmid DNA, otherbacteria that have taken up other DNA,both recombinant and nonrecombinant.
STEP 4 TRANSFORMATION
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STEP 4. TRANSFORMATIONOF LIGATION PRODUCTS
The process of transferring exogenousDNA into cells is call transformation
There are basically two generalmethods for transforming bacteria. Thefirst is a chemical methodutilizingCaCl2 and heat shock to promote DNA
entry into cells. A second method is called
electroporation based on a short pulseof electric charge to facilitate DNAu take.
CHEMICAL TRANSFORMATION
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CHEMICAL TRANSFORMATION
WITH CALCIUM CHLORIDE
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TRANSFORMATION BY
ELECTROPORATION
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STEP 5. GROWTH ON AGARPLATES
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4. Cloning of cells (and foreign genes).
We can plate out the transformed bacteria onsolid nutrient medium containing ampicillin anda sugar called X-gal.
Only bacteria that have the ampicillin-resistanceplasmid will grow.
The X-gal in the medium is used to identify plasmidsthat carry foreign DNA.
Bacteria with plasmids lacking foreign DNA stain bluewhen beta-galactosidase hydrolyzes X-gal.
Bacteria with plasmids containing foreign DNA arewhite because they lack beta-galactosidase.
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5. Identifying cell clones with the rightgene.
In the final step, we will sort through thethousands of bacterial colonies with foreign DNA
to find those containing our gene of interest.One technique, nucleic acid hybridization,
depends on base pairing between our gene anda complementary sequence, a nucleic acid
probe, on another nucleic acid molecule. The sequence of our RNA or DNA probe depends on
knowledge of at least part of the sequence of our gene.
A radioactive or fluorescent tag labels the probe.
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The probe will hydrogen-
bond specifically tocomplementarysingle strands ofthe desired gene.
After denaturation(separating) the DNA
strands in the plasmid,the probe will bindwith its complementarysequence, taggingcolonies with thetargeted gene.
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Because of different details between prokaryotesand eukaryotes, inducing a cloned eukaryotic geneto function in a prokaryotic host can be difficult.
One way around this is to employ an expression vector,a cloning vector containing the requisite prokaryoticpromotor upstream of the restriction site.
The bacterial host will then recognize the promotor andproceed to express the foreign gene that has been linkedto it, including many eukaryotic proteins.
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The presence of introns (the non-coding regions)in eukaryotic genes creates problems forexpressing these genes in bacteria.
To express eukaryotic genes in bacteria, a fully processedmRNA acts as the template for the synthesis of acomplementary strand using reverse transcriptase.
This complementary DNA (cDNA), with a promoter,can be attached to a vector for replication, transcription,and translation inside bacteria.
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ComplementaryDNA is DNAmade in vitro
using mRNA as atemplate and theenzyme reversetranscriptase.
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Molecular biologists can avoid incompatibility problems byusing eukaryotic cells as host for cloning and expressing
eukaryotic genes.
Yeast cells, single-celled fungi, are as easy to grow asbacteria and have plasmids, rare for eukaryotes.
Scientists have constructed yeast artificial chromosomes
(YACs) - an origin site for replication, a centromere, and twotelomeres -with foreign DNA.
These chromosomes behave normally in mitosis and can
carry more DNA than a plasmid.
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Another advantage of eukaryotic hosts isthat they are capable of providing theposttranslational modifications that manyproteins require.
This includes adding carbohydrates or lipids.
For some mammalian proteins, the host mustbe an animal or plant cell to perform the
necessary modifications.
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Many eukaryotic cells can take up DNA from their surroundings, butoften not efficiently.
Several techniques facilitate entry of foreign DNA.
In electroporation, brief electrical pulses create a temporary hole in theplasma membrane through which DNA can enter.
Alternatively, scientists can inject DNA into individual cells usingmicroscopically thin needles.
In a technique used primarily for plants, DNA is attached to microscopic
metal particles and fired into cells with a gun. Once inside the cell, the DNA is incorporated into the cells DNA by natural
genetic recombination.
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In the shotgun cloning approach, a mixtureof fragments from the entire genome isincluded in thousands of differentrecombinant plasmids.
A complete set of recombinant plasmid
clones, each carrying copies of a particularsegment from the initial genome, forms agenomic library.
The library can be saved and used as a source ofother genes or for gene mapping.
4. Cloned genes are stored in DNA libraries
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A more limited kind of gene library can be developed fromcomplementary DNA (cDNA).
During the process of producing cDNA, all mRNAs are converted
to cDNA strands.
This cDNA libraryrepresents that part of a cells genome thatwas transcribed in the starting cells.
This is an advantage if a researcher wants to study the genes
responsible for specialized functions of a particular kind of cell.
By making cDNA libraries from cells of the same type atdifferent times in the life of an organism, one can trace changesin the patterns of gene expression.
cDNA synthesis
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y
TTTTTTTTT
First strand
Nick translation
cDNA library
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EcoR I
Genomic Library
Infect cells
AAAAAA AAAAAA AAAAAAAAAAAA
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AAAAAAAAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAAAAAAAA
AAAAAA
AAAAAA
cDNA synthesis
Infect cellscDNA library
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Genomic library cDNA library
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Genomic library cDNA library
Genomic DNA mRNASource
Species or strains Species or strains
Tissues
Developmental stages
Variation
12k -- 20k 0.2k -- 6kInsert size
Equal Correlate withexpression level
Representation
Only one Expression vs. non
expression
Type
DNA DNA or antibody or
protein
Probe
Gene structure
Infer protein identity
Encoded protein
Infer protein identity
Purpose
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Subcloning of the cDNA insert
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EcoR I
Subcloning of the cDNA insert
+
EcoR I EcoR I
Is there a better way to
subclone the insert?
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Whats the trouble with isolating a gene?
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All genes are made of the same molecule that is chemically quitehomogeneous (DNA all As,Cs, Gs and Ts)
There is a huge amount of genetic material
E. coli>1000 genes (4,600,000 bp)
Humans perhaps 30,000 genes (app. 4,400,000,000 bp)
Single genes only make a tiny fraction of the whole genome
In E. coli, each gene is app. 0.05 % of the genome
In humans, each gene is app. .00005% of the genome
Finding a gene is not like like looking for a needle in a haystack, it is morelike looking for one particular piece of hay in a haystack! In this case allthe hay looks the same, and you can only tell the difference by analyzing
the chemistry of each piece!
Whats the trouble with isolating a gene?
Break the genome into more manageable pieces
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Restriction enzymes - isolated from bacteria probably involved inprotecting the cell from viral foreign DNA
Example: EcoRI from E. coli
Cleaves six base pair siteE. coliDNA is protected by acorresponding DNA methylase that adds methyl groups to the centralbase pairs and prevents cleavage
These enzyme are purified and used as tools in vitro
Will cleave DNA at any GAATTC sequence - every genome has aspecific pattern of GAATTC sequences that occurs at random, but is
always the same
Break the genome into more manageable pieces
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Properties of plasmidcloning vector
1. Small2. Stable in the chosen
host usually E. coli3. High copy number4. Easy to purifiy5. Can accommodate
foriegn DNA
6. Single cloning sites7. Selectable markerantibiotic resistance
8. Easily introduced intohost (transformation ortransduction
How do you stably maintain and replicate a foreignDNA fragment? by hitching a ride on a stablereplicon
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pBR322, cut atits singleEco RI site
EcoRIEcoRI foreignDNA
EcoRIEcoRI
DNAligase
recombinantDNA
NNNGAATTNNNC
EcoRI end on insert
CNNNTTAAGNNN
EcoRI end on vector
Sticky ends
NNNGAATTCNNNNNNCTTAAGNNN
Fusion site
New Eco RI site
Simple fusion of different DNA fragments
a-complementation relies on modular structure of b-
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a-complementation
LacZ+ - blue colony
LacZ- - while colony
-if you interrupt the lacZgene, the colony is white
a complementation relies on modular structure of bgalactosidase
- basic idea is often used with cloning vectors called insertional inactivation
How does a-
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How does acomplementationwork?
It all comes down to b-galactosidase
Certain strains supply theb-Gal fragment
When a is supplied intrans, this allows b-Gal to
function
One major issue is the size of the insert DNA
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One major issue is the size of the insert DNA
The larger the fragment, the more of the source genome can berepresented on one plasmid
For a gene library, we can easily calculate the number of individualrecombinant molecules required to represent an entire genome
N=[ln (1-P)]/[ln (1-f)]Pprobability of complete coverageN number of individual clones requiredf proportion of genome in averagefragment
N=[ln .01]/[ln 0.99999886]
= -4.6/-1.14 x 10-7
= 40,350,877 individuals
For the human genome and astandard plasmid
P 99% or 0.99 confidencef5 kb/4,400,000 kb = 1.14 x 10-6
Plasmid vectorsstable inserts of 5 kb
B t i h t d t l i t
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larger inserts than plasmids introduced by phage infection of cells
20 35 kb inserts
Bacteriophage vectors can acommodate larger inserts
N=[ln .01]/[ln 0.99999205]= -4.6/-7.95 x 10-6
= 578, 616 individuals
For the human genome and astandard plasmid
P 99% or 0.99 confidencef 35 kb/4,400,000 kb = 7.95 x 10-6
A big improvement!
Yeast artificial chromsomes (YACs) and
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Can accomodate from 300 500 kb of DNA great forlarge genomes
Yeast artificial chromsomes (YACs) andbacterial artificial chromsomes (BACs)
N=[ln .01]/[ln 0.999886]= -4.6/-1.14 x 10-4
= 40,350 individuals
For the human genome and astandard plasmid
P 99% or 0.99 confidencef 500 kb/4,400,000 kb = 1.14 x 10-4
A even bigger improvement!
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Whats a YAC?Yeast artificial
chromsome
self-replicatingvector that can bemaintained in yeast
Can accommodatelarge insertfragments
Reeves et al., 1992, Methods Enzymol. 216:584-603
What are BACs?Derived from the F plasmid of E. coli
very stably maintained
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What are BACs?bacterial artificialchromsomes
- very low copy numbervectors that can accomodate
huge inserts
Shizuya et al, 1992, PNAS 89:9794-8797
-very stably maintained- 1-2 copies per cell (strict copynumber contro)
Human
BACSource Plate
Caltech &
GDB
Average
InsertAvailable Cloning
NHGRI-
DOEConstructedA wide
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BAC
librariesmaterial numbers
GDB
Names
Insert
Sizefrom Site
DOE
approvalby
A987SK
cells
1-1,000 (96-
well)
CIT978SK-1A1 to
1000H12115 kb Caltech HindIII
TatianaSlepak,
ValeriaMancino
B 987SKcells
1-194 (384-well)
CIT978SK-1001A1 to1194P24
120 kb ResearchGenetics
HindIII Yu-LingSheng
C
humansperm
(HSP)
195-879 (384-well)
CIT-HSP-1195A1 to
1900P24
125 kbResearchGenetics
HindIIIYu-LingSheng
D1
humansperm
(HSP)
2,001-2,423
(384-well)
129 kb
Caltech,
GenomeSystems,
ResearchGenetics
HindIII ApprovedYu-Ling
Sheng, Yu-
Jiun Chen
human
sperm(HSP)
2,501-2,565(384-well)
202 kb
Caltech,Genome
Systems,ResearchGenetics
EcoRI Approved
humansperm
(HSP)
2,566-2,671
(384-well)182 kb
Research
GeneticsEcoRI Approved
human
sperm(HSP)
3,000-3,253
(384-well)142 kb
Research
GeneticsEcoRI Approved
D2
humansperm(HSP)
3,254-transforamtion
on-going(384 well)
CIT-HSP-2000A1
and up
166 kb EcoRI Approved
SangdunChoi
A widerange ofBAC
libraries arenowavailable forthe humangenome aswell asother large
genomes
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