CH 17 Recombinant DNA and Gene Cloning

Alot of restriction enzymes, only made by bacteria. NOt every bacterial cell makes restriction enzyme, but many have atleast one strain.

-5 different Eco enzymes (1-5) Each cut at different site in sequence -Blunt ends: DNA fragments with ds ends, hard to clone -Some enzymes have recognition sequences of 6 bases, or 4 bases, the length of the sequence influences how often they cut -Different enzymes give different base sequence size -4 base cutter is most frequently used: cuts it into "gene" size, exact size just depends on the sequence being used. Avg: 4000 bases.

Cloning by PCR- Polymerase Chain Reaction

-Allows amplification of specific DNA sequences -Used to clone genes without making a library -Wide range of applications in genetics, molecular biology, evolution, medicine, criminal cases.

Analysis of Cloned Genes - restriction mapping

-Can map a gene once it is cloned -Cloned DNA ran on gel after being cut with restriction enzymes -Cloned DNA into a plasmid -Cut plasmid w. different restriction enzymes; use this to make restriction map and see where they are cut within the plasmid -2 size markers indicates the enzyme cuts twice -The size markers indicate hoe far the cuts are from each other

Southern Blot

-DNA from organism cut with restriction enzymes, ran on agarose gel, probed for specific sequence, used to study STRUCTURE of a chromosome -Can find which clones in a library contain a given DNA sequence

Process of Southern Blotting

-DNA samples cut w/ restriction enzymes, loaded on gel for electrophoresis -DNA is denatured with alkaline fragments to ssDNA. -Gel is overlayed with DNA binding membrane (usually nylon), and transfered to wic sponge in buffer solution. -Buffer is sucked up through the gel, transferring DNA to membrane -Membrane filter places in heat sealed bag with solution containing ss radioactive DNA probe -Membrane is washed to remove unbound probe, dried, xray film applied -Hybridize the membrane using a radioactive probe (cloned DNA, made radioactive using pol1) -Pieces of DNA bounds are complimentary to the probe Autoradiogram--> all size markers show because they are all radioactive, only mands that hybridize with probe will show up in other lanes.

2 Types of Gene Libraries

-Genomic DNA libraries: cloned fragments of DNA isolated from chromosomes; represents all DNA sequences. Used for cloning regulatory sequences (promoters), studying genome organization, sequencing of whole genomes, if you want to find introns. -cDNA libraries: cloned DNA copies of mRNA isolated from cells or tissues, used for isolating genes expressed in certain cells and tissues under certain conditions. DNA in a genomic library represnet coding and noncoding genes, cDNA only contains expressed genes.

Can also use northern blots to study splicing reactions, can infer that splicing is done differently in brain than in other tissues.

-Has different amount of RNA -Tells us about alternative splicing, size of mRNA transcript, and estimates relative transcriptional activity of a gene.

Disadvantages of PCR

-PCR not necessarily specific for finding the intended sequence; Primers are hard to design, make sure primers dont hit multiple sites in genome. -Primers and annealing temperatures determine the specificity of PCR; temp really important. -Make sure primers don't bind to off target sites. If they do, after 1 round of replication it will bind perfectly to nonperfect site. -Anneal temperature based on MP of primers, run 3 degrees cooler than MP. -You can amplify something unintended. -Short vs. Long PCR--> not necessarily accurate, cannot copy long pieces of DNA with taq polymerase bc of error rate- to get around this they do a long PCR with an enzyme with proof reading function. -Taq Poly lacks 3' to 5' exonuclease activity- proof reading function; enzymes will introduce errors into DNA that you're copying - when it puts in wrong base it stops replicating and reaction terminates.

PCR is a rapid method of DNA cloning > eliminates the use for host cells

-PCR used to amplify target DNA sequences that are initially present in small quantities. -ds target DNA + DNApoly + 4 DNA bases (A/T/G/C) are added to test tube. -some info must be known about target DNA in order to synthesize 2 ss oligonucleotide primers. -primers bind to complementary DNA after they have been denatured, then they are extended by DNA poly at the 3' end to make a clone -each cycle doubles the number of DNA molecules in the reactioni -Specific DNA polymerase is used bc not all of them can handle the temp change from annealing to cooling- Taq polymerase -DNA used can come from many samples, even used for forensics like hair samples and dried blood.

Restriction Enzymes

-Restiction endonuclease: recognizes specific restiriction site and cuts DNA in a sequence specific manner, usually in the center -Recognition sites are PALINDROMIC sequences: nucleotide sequence reads the same on both strands of the DNA when read in the 5' to 3' direction. -Complimentary ends, are "sticky ends" and naturally stick together through complimentary base pairing, used for reassociation (different pieces from different sources) -EcoRI have sticky ends, the vector must be cut by the same restriction enzyme

Different restriction enzymes cut different DNA sequences

-Restriction enzymes come from multiple strains of bacteria -Bacteria that make restriction enzymes also make corresponding methylase. This corresponding methylase stops the bacteria from cutting its own DNA at sites that the restriction enzyme would otherwise recognize in the bacteira.

PCR Uses DNA Polymerase to copy DNA in the test tube repeated rounds of replication

-Specificity of PCR depends on choosing the right primers and the annealing temperature used -DNA replication in testtube 1. Denature to ssDNA 2. oligonucleotide primers flank pieces of DNA we want to target 3. Cool DNA down to temps that allow primers to bind 4. DNA polymerase copies, go back and do it again.

Several Types of "Vectors" are used in cloning, they differ in the size of the "inserts" they can carry.

-Standard Plasmids: inserts <10kbp -Bacteriophage Vectors: inserts 10-50kbp -BAC Vectors: inserts 100kbp **different vectors carry different size pieces of dna, making them useful for different purposes. -Standard bacterial plasmids can hold about 10,000 bases of DNA -Viruses: lambda, can hold a piece of DNA up to about 50,000 basepairs, typical size 25,000. This type of virus is lysogenic; it enters bacterial cell and becomes dormant. If we want to use bacteriophage as a vector, we have to isolate the end pieces and disregard the genes in the middle. Use these ends to clone DNA into it and the virus will package it. The f plasmid: only part is needed is the part for replication **lysogeny- viral DNA is integrated into bacterial chromosome, so every time the chromosome is replicated the viral DNA is replicated and passed down.

Why do bacteria have restriction enzymes? Whats interesting ab it?

-These are sequences that cut DNA so youd expect it to cut its own DNA which would be lethal, but it makes restriction enzymes and a corresponding methlyase at the same time, which methylates the other stands, blocks cutting. -This doesnt happen in humans, so these enzymes are for bacteria's immune system. -Foreign DNA is moved around and if the cell has the restriction enzyme it can cut the foreign DNA up and protect itself from being gentically modified. ^We get rid of this function in the lab so DNA can be cloned.

Limitations of PCR

-information about DNA is needed to make primers -minor contaminations of DNA can cause problems for accuracy (ex: skin cells from contaminate samples from crime scene) -Usually DNA poly in PCR only extends primers for short distances, not to the end of the strand, So PCR is usually used to amplify relatively short pieces of DNA.

Next Gen Sequencing

-revolution of dna sequencing: sequencing made cheaper and faster -goal is to make sequencing genomes cheaper -approach based on PCR type reactions -era of personalized medicine based on gene sequences, you can target specific spots in genome to obtain background medical information

Oligonucleotide synthesis

-ssDNA sequences (up to 40-50 bases) are easily synthesized. -Synthetic reactions: add bases one at a time to be made into short sequences. -short, ssDNA fragments can be used as a DNA PROBE for alleles that differ by even a single nucleotide. Hybridization only to complimentary base sequence, not even to sequence off by 1 base.

Process of PCR

-start with ds DNA to be cloned in test tube with primer (present in excess), DNA polymerase, and 4 DNA bases (A/T/G/C) -first denature the DNA (heat 92-95deg) -primers anneal to the single stranded DNA (when its cooled to 45-65 deg) -the primers are extended by DNA polymerase (Taq) at the 3' end, then temp is raised to 65-75deg -end of first cycle; number of DNA molecules have doubled

Northern Blot of Human RNA from different organisms: Probed with cloned FMR1 gene (mutations in this gene result in mental retardation)

-take RNA from different cells, run it out on agarose gel, and transfer to a membrane paper, and run it with ssDNA probe derived from a cloned copy of FMR1 gene (fragile x) RNA from different tissues. -Northern blot tells us where genes are expressed and where. -The mRNA that corresponds to FMR1 is 4.4 bases long, and that there is more in the brain, placenta, etc and very little in the liver. -Darker bands mean greater expression -Bigger bands mean more RNA that correspond. -In the heart the message is small, may be due to alternative splicing.

Steps in Cloning a Gene

1. Isolate DNA from the organism 2. Cut DNA with restriction enzymes 3. Insert DNA fragments into a "vector" (plasmid) 4. Introduce recombinant DNA into bacterial host cell (E.coli) for replication 5.Grow the host and reisolate recombinant DNA clones **to distinguish host cells carrying plasmids with dna inserts, the vector should have a selectable marker gene- usually an antibiotic gene or an enzyme absent from host cell, when plated on this antibiotic it will be obvious to see host cells.

Strategies for Screening Libraries

1. Probing using an already cloned gene 2. Isolate the protein, sequence it, can now predict sequence of protein, able to predict end of nucleic acid sequence, and make a synthetic oligonucleotide 3. Expression libraries: antibody detection used

Screening a gene library

Can be done by DNA hybirdization using a cloned gene as the "probe" -the probe is a DNA or RNA sequence that is complementary to the target gene/ sequence to be identified in the library. 1. colonies of the library are overlaid with a DNA binding membrane such as nylon> this transfers the pattern of bacterial colonies from the plate to the membrane. 2. colonies are transferred to the membrane, then lysed and denatured with NaOH 3. Membrane is placed in a heat sealed bag with a solution containing the labeled probe; the probe hybridizes with complementary DNA from colonies. 4. Membrane is removed from bag and rinsed to remove excess probe, then dried; xray film is placed over membrane> hybirdization of a clone to colony shows up by a spot on xray film. 5. colonies containing the inserted probe are identified from the orientation of the spots on the original plate, and removed. 6. cells that hybridized with the probe are transferred to a medium for growth and further analysis

Gene Library

Cloning DNA yields a "gene library"-- collection of clones that contain all the DNA sequences of an organisms genome ~1million different pieces of DNA. When you throw the whole genome into plasmids, you get a lot of colonies to look at before you find the specifc gene you want. Major challange: how to identify the specific clone that has the gene you want? Screening libraries "finding the needle in the haystack"

Analysis of Cloned Genes- DNA Sequencing

Dideoxynucleotide sequencing- has an -H and not an -OH at the 3' end. -block further DNA replication, chain terminating There's no where to form the phosphodiester bond to the next nucleotide - YOU NEED ..... -DNA polymerase -DNA template -primer -dATP,dGTP,dCTP,dTTP (each has different color) -small amount of ddNTP that are florecently labeled DNA replication in a testtube 1.primer is annealed to DNA template 2.Add rxn mixture together, primer extension and chain termination- ddNTP is inserted at every location, terminating the chain so that each strand differs in length by 1 nucleotide. (if regular G was placed, replication would continue). Many copies of template are in the test tube. -->Results in cluster of fragments each ending in different base. 3. products are added to single lane on a capillary gel electrophoresis, and the bands are read by a detector system--> computer converts this into a sequence. can determine single base differences in a molecule, results in series of peaks.. Products are made single stranded. 4. the sequence is found by the extension of the primer, and is read from the new strand, not template strand.

DNA from an organism and the vector DNA is cut by the same restriction enzyme, their complementary tails base pair with each other, then the gaps are sealed with DNA ligase --> This makes Recombinant DNA!!

Done in a test tube at low temperature, this is necessary for hydrogen bonds to stabilize and stick together. Ligase seals the phosphodiester bonds and now they're bonded from 2 different sources.

Restriction enzyme mapping of plasmids **** look at slides**

EX: EcoRI cuts twice at 7 and 3, the distance between the 2 cuts will add up to 7 in 1 direction, and adds to 3 in the other direction. HindIII cuts once so it doesnt matter where it starts PStI cuts once, and 7 is digested

Retroviruses

Ex: HIV RNA viruses that replicate by turning their genome into DNA and inserting it into other genomes.

Analysis of Cloned Genes

How genes are put together inside cell/chromosome Also used to measure expression *Southern blot: detection of DNA sequences on a gel, used to study gene structure *Northern blot: detection of RNA sequences on a gel used to study the amount of mRNA present (transcription)

Genomic DNA cut with different restriction enzymes and then run on different lanes, DNA stained with ethidomine bromide- Southern Blot

Individual DNA bands are not usually seen because there are so many different fragments -No bands shown, only smear shown because there are really alot of bands, small bands shown are repeated staellite sequences. -Bands containing DNA sequences complimentary to probe --> SHOWS hybridization. *Sizer of bands proportional to size of restriction site that cut around the sequence

Recombinant DNA

Joining of DNA from different organisms in the test tube -isolation of individual genes (Gene Cloning) -study of gene function and regulation of expression -cloned genes can be sequenced and their base sequences can be modified -Transgenic organisms: insertion of cloned genes into animals plants or bacteria "GENETIC ENGINEERING"

Vectors

Plamids -origin of DNA replication -antibiotic resistance gene -unique restriction sites *Size of the recombinant fragment is limited to ~10kb Vectors are genetically modified bacterial plasmids used for cloning -extrachomosomal, dsDNA that replicate independantly from the chromosomes within the bacterial cells. Basic features include: ability to replicate (origin of replication), antibiotic resistant genes (so you can introduce the plasmid without DNA cells recognizing it) and specific site on plasmid which was modified and the restriction enzyme cuts here only once, the plasmid opens up and accepts insert. -the plasmid vector is removed from the host cell and cut by restriction enzyme, DNA to be cloned is cut with the same restriction enzyme, and then these 2 pieces are ligated and introduced into bacterial host cells by TRANSFORMATION

Multiple cloning site

Plasmid vectors have this which allows them to have many restriction sites for many restriction enzymes; allows scientists to clone a range of different fragments generated from many common restriction enzymes.

Northern Blot

RNA from specific cell/tissue, separated by electrophoresis, transferred to membrane to me probed, ran on gel, probed for specific sequence (probes for the presence of mRNA complementary to cloned gene) *Used to study GENE EXPRESSION/ how much mRNA is present

Lambda phage

Restriction enzymes remove the central gene cluster, leaving the 2 lambda arms on end and opening in middle. Insertion and ligation of foreign DNA is done, this is IN VITRO packages into phage head > ** recombinant viral particle is able to infect bacterial host cell and replicate to form plaques that carry the cloned foreign insert.

White colonies carry a recombinant DNA plasmid, Blue colonies are just plasmid alone.

The cells making beta G turn blue, if they don't, they're white. The white colonies contain a plasmid with an insertion ability. The blue ones have only the plasmid. -Original plasmid has lac-z gene to make x-gal and form blue colonies > multiple cloning site is cut > DNA cut with same restriction enzyme, added and ligased to plasmid > DNA frag inserted anywhere in multiple cloning site, the lac Z gene within the multiple cloning site is disrupted and cannot make beta G > recombinant plasmid cannot metabolize x-gal and white colonies will form.

Output of a DNA sequencing reaction using fluroecently labeled bases

The fluorecent tags on the detector and signal system: as you get to longer pieces, the peaks go down and the widths become wider--> harder to distinguish between bases.l ** Technology came out of Human genome project.

Southern Blot of DNA from 4 species: human, mouse, hamster, and cow -Cut with EcoRI or HindIII or PstI -Probed with a fragment of the human cystic fibrosis gene

This is called a zoo block - they use animal DNA and probed it with the human cystic fibrosis gene to see if it would hybridize - aka is their DNA complementary to our DNA sequence for CF gene? -Piece of DNA we used to prove, has ecoRI site on either side, pstI about 9000 bases. -The cow showed some hybridization to the human gene, yet not in the same places as the human -The mouse and hamster don't have any hybridization- showing a bigger evolutionaly difference between hamsters/mouse vs us+cows. -Mouse- sequences arent the same, hybridization doesn't work well

DNA libraries

Used to know what DNA sequences are being cloned- genomic libraries and cDNA libraries insert genome into vectors, and cloning them in host cells. ** Once they are both cloned, they are probed for something specific.

Glo Fish and Glo Pigs

engineered to express the gene for green fluorescent protein cloned from jelly fish

Restriction maps

establishes the number of, order of, and distances between restriction sites along a cloned segment of DNA Provides information about the length of the cloned gene and the location of restriction sites within the clone

cDNA

the DNA copies are made from mRNA molecules of a cell population and therefore represent the genes expressed in the cells; cDNA is "complementary DNA" made from mRNA

Each bacterial colony is a clone, carrying a specific cloned DNA sequence

the whole thing plated on the medium is a colony, and each are a clone of some specific DNA seqeuce

cDNA synthesis

uses reverse transcriptase to make a DNA copy of mRNA-- the sources of this is retroviruses. 1. Isolate total RNA from cell, mix mRNAs with ss oligo-dT primer (TTTT), these base pair with polyA tail. 2. reverse transcriptase extends the oligo-dT primer amd makes a complementary DNA copy of the mRNA 3. the RNA part of the hybrid (because now you have 1 strand DNA, and 1 strand RNA), is partially digested by RNAse-H, the 3 ends of the remaining RNA serves as primers for DNA poly 1, to make the second strand of DNA, filling in spaces. 4. The opposing strand of DNA is made with DNA poly1. The primer till chug along and cut out RNA and replace with DNA. Add ligase to make the final ds cDNA molecule. This DNA piece now corresponds with the messanger. ***Replication starts at the primer site for reverse transcriptase-- polyA tail 3' end Must manipulate more steps to get DNA into plamid