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DNA Technology

-->Coverts scientist knowledge of the human genome into better ways to diagnose & treat diseases
-->Helps to understand the molecular basis of diseases
-->Can be used to produce human proteins in abundance (Ex. Insulin)
-->Can be used to make proteins for vaccinations (Ex. Hep B)
-->Helps to diagnose diseases & predict risk
-->Also used in **forensic medicine**

List of DNA Technologies

1)DNA Cloning

Restriction Endonucleases

-->Enzymes that cleave dsDNA very selectively at PALINDROMIC SEQUENCES of 4 to 8 nucleotides
1)Represents DNA whose 5' --> 3' sequence is identical on both strands
-->Produces **restriction fragments** that depends on the cleavage specificity of the enzyme
-->Most RE's cut one or two base pairs away from the symmetry axis in both strands, resulting in ds DNA with **short single-stranded ends**
-->B/c the ss overhangs are COMPLEMENTARY TO EACH OTHER, every restriction frag. can ANNEAL (or base pair) with any other restriction fragment produced by the SAME restriction enzyme
1)Once annealed, the restriction fragments can be linked covalently by **DNA Ligase**
2)Property makes RE's ideal for **recombinant DNA technology**(The cutting & joining of DNA in a test tube)

If the Restriction Endonuclease (RE)recognizes a specific sequence of 4 (8) bases

-->The restriction fragment that it will produce will be, on average, 256 (65,536) base pair segments in LENGTH 4^4 (4^8)

***Bacterial Enzymes***

-->Can be used as RE's as DEFENSE against DNA Viruses
-->MOA:
1)While some suspetible sites in the bacterial genome are protected by methylation (due to a methylase accompying the use of the RE), the unmethylated DNA of viral intruder DNA is CLEVED

Panlindrome

-->A region of the double helix DNA that contains the SAME nucleotide sequence on the 2 strands if each is read in the 5' -->3' direction
-

Blunt vs. Sticky Ends

1)Blunt Ends:
-->The production of restriction fragments that are **double-stranded**(so they cannot form hydrogen bonds with each other
-->Produced by **Hpal**
2)Sticky Ends
-->The production of restriction fragments that are single-stranded (that are complementary to each other)
-->Produced by **BamHI**

Examples of Restriction Endonucleases & where exactly they cut

1)E.CorI cuts at (5') G *** AATTTC (3') {**Must know EXACT sequence 5' to 3'}

Requirements of DNA Cloning

-->**DOES NOT require the integration of the DNA into the bacterial genome**
-->A CLONING VECTOR (Insertion of foreign DNA in a self-replicating entity)(Cloning Vector can be a plasmid or bacteriophage)(Requires the covalent joining of the foreign DNA with the vector DNA)

The procedure for DNA Cloning

-->Contains an R-factor plasmid carrying resistance genes for TETRACYCLINE & AMPILLICIN
-->Plasmid & human DNA are cleaved with the same highly selective restriction endonuclease
-->The circular plasmid DNA is cleaved at only one site, while the DNA is fragmented into pieces of many thousands of base pairs
-->Plasmid & human DNA are MIXED in the test tube, and their cohesive ends anneal spontaneously
-->DNA ligase is ADDED, and the PLASMID DNA & HUMAN DNA are covalently linked in a circle
-->The recombinant plasmids are spirited into the bacteria via TRANSFORMATION
-->Recombinant clones are selected on the bases on growth on an ampicillin-containing & nongrowth on a tetracycline-containing medium
--
-->Procedure generates ***millions of bacteria with recombinant plasmids
-->A large collection of transformed bacteria (each containing a random piece of human genomic DNA)
-->The host cells with the recombinant vector are selected for using the antibiotic resistance genes
1)For Ex. if the foreign gene is inserted into the tet(r) gene, **IT IS INACTIVATED** (i.e. the host cell becomes SENSITIVE TO TRETRACYCLINE, but RESISTANT to ampicillin)

Problems with sticky end ligation

-->Although its EASIER, it can be problematic b/c:
1)sticky ends bind TO EACH OTHER instead of places within the vector-no-net gain of DNA
2)May not be available in a convenient
3)Heterogeneous tandem can form when sticky ends anneal

Features of Blunt End Restriction Enzymes

-->Can be ligated DIRECTLY but not DIRECTIONALLY

Enzymes Used for Recombinant DNA

Features of Vectors

-->Plasmids are useful vectors - circular dsDNA molecules found extrachromosomally
-->Contain origin of replication
-->Genes for antibiotic resistance-useful for selecting for recombinant DNA
-->Must have many palindromic sites for REs to recognize
-->Can use viral vectors such as viral chromosome (phage) or yeast artificial chromosome ( YAC)

Plasmids

-->Carrier molecules that contain gene of interest and can be inserted into bacterial cell and replicated
-->Must have same recognition site for Restriction Endonucleases as gene of interest
-->Ligase attaches foreign gene into plasmid by making covalent bonds

Features of Selection for Recombinant DNA

-->Involves the use of the antibiotic resistance gene or genes in the cloning vector, which are referred to as SELECTABLE MARKERS
-->Ex. of the tretracycline resistance gene being disrupted by the insertion of foreign DNA, whereas the amipicillin resistance gene is still intact
1)Thus, properly transformed bacteria can grow in the presence of of ampicillin, BUT NOT TETRACYCLINE
-->The transformed bacteria can be transferred to an AGAR PLATE, where each grows into a visible colony
1)Each colony consists of a CLONE of genetically identical bacteria that carry the same insert of foreign DNA
--
NOTE: The **X-gal** method (gene appears blue when grown on a medium that contains the substrate analog X-gal) is MORE EFFICIENT than the agar plate methond

Selection for ***Recombinant DNA***

-->To select for cells + plasmid + gene of interest, must stamp and plate on media containing another antibiotic eg. Ampicillin and tetracycline
-->**Gene of interest** will have interfered with resistance of plasmid to that antibiotic, therefore cells with recombinant DNA will die
-->**Based on location of DEAD colonies on agar plate**, we can determine recombinant colonies on original agar plate

Reporter Genes of Plasmids

-->Genes that encode a protein that can be used to identify correct colonies
-->Eg. **Β-galactosidase** that causes bacteria expressing its gene (lacZ) to turn blue when grown in media containing its substrate analog X-Gal

Isolating Genes of Interest using Complementary Probes

-->Press nitrocellulose paper onto transformed colonies
-->Add ssDNA probe to hybridize with this gene
1) probe is complemetary sequence so need to know a little of the sequence of the gene
-->To find colony with gene, we tag probe **32P** and incubate bacteria with probe
-->Then take radiograph and trace back to original colony

Molecular Probe

-->A **single-stranded DNA or RNA** that is complementary to the target DNA

Mamalian Viral Vectors

--->Vectors that are based upon eukaryotic viruses composed of DNA or RNA genomes
-->Some vectors are Adenoviral (Ad), or adenovirus-associated viral(AAV) DNA-based, and retroviral (RNA-based) genomes
-->**Used because the efficiently infect a wide range of different cell types**
-->Used for gene therapy

2 Types of DNA Libraries

1)Genomic & ComplementaryDNA Libraries

Genomic Libraries

-->Collection of the ENTRIRE HUMAN DNA (Genes + Junk DNA)

cDNA Libaries

-->Represents collection of EXPRESSED DNA of a cell type or tissue (i.e. genes ONLY)

Genomic Library

-->Represents a large collection of transformed bacteria, each containing a randome piece of human genomic DNA
-->Ex. ***Taql (4 base cutter)
-->In the genomic library, the DNA is cloned into vectors that accept very large fragment (Ex. BAC, YAC & P1)

Expression Vectors

-->Represents the effective transcription & translation of **CLONED DNA**
-->Especially useful to help genetically engineer bacteria
-->Requirements of the use of the expression vector:
1)Only cDNA can be expressed in bacteria
2)The coding sequence (cDNA) must be joined to a strong bacaterial promoter
3)The 5'-untranslated region of the transcript must contain a Shine-Dalgarno sequence (required for the initiation of translation)
-->Represents the vector where the protein coded by the gene is actually SYNTHESIZED (used to detect cDNA mol. in libraries)

CDNA Libary

--)>Is a collection of all the expressed DNA in a cell, starting from **mRNA** (rather than genomic DNA in genomic DNA libraries)
(i.e. Proteins --> mRNA --> cDNA)
1)RNA is hydrolyzed so that dsDNS forms & is ready for insertion into a vector
-->DOES NOT contain promoters & enhancers b/c it is made from mRNA that has ALREADY BEEN TRANSCRIBED
-->***Use a oligo(dT) primer

Mechanism of formation of cDNA library (from mRNA)

1)mRNA templated is annealed to synthetic oligionucleotide (oligio dT) primer
2)**Reverse Transcriptase & dNTPs** yield a complementary DNA strand
3)mRNA is degraded with alkali
4)DNA ploymerase I & dNTPs yields a ds-DNA

***See slide 24 of TA session to compare genomic libary & cDNA libary

--

Screening of Libraries

-->A step-wise method to identify a bacterial colony carrying a particular DNA clone:
1)A bacteria carrying vectors with inserted DNA fragments are placed on a Petri dish (by pressing a piece of absorbent paper against the surface of the dish
2)The replica is treated with alkali (to lyse the cells & separate the plasmid DNA into single strands)
3)The paper is then hybridized to a highly radiactive DNA probe (Ex. I (125) )
-->The bacterial colonies that have bound to the probe are identified using autoradiography
4)Living cells containing the plasmid can then be isolated from the original dish

Human Genome Project

-->Application of Genomic library
-->Cut genome using Res, then clone fragments into vectors
-->Fragments are re-isolated from recombinant vectors and sequenced
-->Useful for identifying specific mutations associated with diseases
-->Identifying recognition sites for REs (Restriction map) to be used as **RFLP markers for genetic diseases**
Introns, regulatory sequences, enhancers, promoters, response elements can be identified

Largest % of the Human Genome Project

-->Contains Tranposons (45%)

Features of cDNA cloning

-->Theraputic agents such as the production of: 1)eg. Human insulin ( Diabetes mellitus), HBsAg (Vaccine for Hepatitis) and factor VIII (Hemophilia)
-->Gene therapy in patients with genetic deficiencies
-->Produce transgenic mice and knockout mice

Gene Therapy

-->Developed for the treatment of genetic disease & some non-genetic conditions
-->This strategy DOES NOT manipulate the germiline
-->It only replaces nonfunctional gene with normal gene **ONLY IN SOMATIC CELLS**
1)Necessary so that the gene is not transferred to the OFFSPRING
-->Methods used to facilitate the uptake:
1)Use of viral vectors (retrovirus; adenovirus)
2)Use of lipid vector (**Liposome**)

Ex-vivo vs In vivo gene therapy (see slide #29 of TA session)

-->Note:
1)Ex vivo gene therapy is more common than in vivo gene therapy

Problems with retroviral vector (ex. RNA viruses)

-->Can cause random integration into the chromosome (maybe next to a proto-oncogene, causing cancer)
-->Used in SCID (Severe Combined Immunodeficiency)

Problems with Adenovirus Vector (Ex. ***DNA Virus***)

-->The therapeutic gene is NOT integrated into the host chromosome, so you may need REPEATED treatments
***Used in Cystic Fibrosis

Retrovirus vs. Adenovirus

***See slide #32

Transgenic Animals

-->The introduction of foreign genes into animials **germ-line cells**
-->When the trangene is introduced into the feritized ova or embryonic stem cell, it is NOT differentiated
1)Only once the eggs are put in foster mother & offspring will contain foreign gene in ALL CELLS including germ cells
-->Used to study the dominant effects of the genes

2 major methods to produce transgenic animals

1)Embryonic Stem Cell Method
2)Pronucleus Method

Embryonic Stem Cell Method

-Insert gene into vector DNA with promoter and enhancer sequences so gene can be expressed in cells
-Allow vector DNA to insert into host cells in culture
Select for successfully transformed cells
Inject these cells into inner cell mass of mouse blastocyst
-Transfer embryo into pseudopregnant mouse (mated with vasectomized male)
-Test offspring = 10-20% will be heterozygous for the gene
-**Mating of 2 heterozygous** mice so 1 in 4 will be homozygous for the transgene
-Mating these homozygous mice will produce

Pronucleus Method

-Insert gene into vector DNA with promoter and enhancer sequences so gene can be expressed in cells
-Inject male pronucleus with DNA
-Allow male pronucleus to fuse with female egg to form diploid zygote nucleus
-Allow zygote to divide to form a 2 cell embryo
Implant embryo in pseudopregnant foster mother

Features of Knockout MIce

-->Deleting certain genes in mice to allow us to study the effects of the absence of the protein product of that deleted gene
-->Knockout mice can be **unaffected* sometimes because mouse genomes have sufficient *redundancy** to compensate for a missing pair of alleles
-->Most genes are pleiotropic (1 gene has more than 1 effect on the individual's phenotype)

Conditional Knockouts

-->Process where a particular gene can get knocked out in only one type of cell
-->Eg. The Cre/loxP system
1)LoxP site is a 34-base pair palindromic sequence that is recognized by the **Cre recombinase**
2)Cre enzyme in bacteriophage cuts viral DNA flanked by 2 loxP sequences and the remaining DNA is ligated together
-->Mice can be made transgenic for a target gene that is flanked by **loxP sequences** as well as a Cre gene
-->When transcription is turned on, the Cre protein will remove the target gene thus generating a knockout
-->All other cells will lack transcription factors needed to bind to the Cre promoter so the target gene remains intact
(i.e. ***the Cre recombinase does not harm the normal DNA b/c the genomic DNA DOES NOT contain any lox P sites)

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