an introduction to ant dna technology

7/31/2019 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


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


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

an introduction to ant dna technology

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