1 Cloning DNA A reasonable question is how did we get the 20kb fragment of DNA in the first place? To understand the origin of the fragment we must address the issue of: The construction of Recombinant DNA molecules or cloning of DNA molecules Recombinant DNA is generated through cutting and pasting of DNA to produce novel sequence arrangements

2 Recombinant DNA Restriction enzymes such as EcoRI produce staggered cuts leaving short single-stranded tails at the ends of the fragment. These “cohesive or sticky” ends allow joining of different DNA fragments

3 Plasmids Plasmids are naturally occurring circular pieces of DNA in E. coli

4 Ligation The EcoRI linearized plasmid DNA is mixed with human EcoRI digested DNA The sticky ends hybridize and anneal and a recombinant plasmid is generated

Plasmid propagation The plasmid DNA can replicate in bacteria and therefore many copies of the plasmid will be made. The human DNA fragment in the plasmid will also multiply along with the plasmid DNA. Normally a gene is present as 2 copies in a cell. If the gene is 3000bp long there are 6x10 3 bp in a total of 6x10 9 bp of the human genome Once ligated into a plasmid, unlimited copies of a single gene can be produced.The process of amplifying and isolating the human DNA fragment is called DNA cloning.

6 Inter-species Gene transfer Human Cell is cf-/cf- It becomes CF+ after transfection CF gene on a plasmid in E.coli Isolate Plasmid Transfect human cell with CF plasmid

7 Plasmids Small circular autonomously replicating extrachromosomal DNA Bacterial genome (5000kb) Plasmid DNA (3kb) Modified plasmids, called cloning vectors Are used by molecular biologists to isolate Large quantities of a given DNA fragment Plasmids used for cloning share three properties Unique restriction site Antibiotic resistance Origin of replication

Plasmid elements Origin of replication: This is a DNA element that allows the plasmid to be replicated and duplicated in bacteria. Each time the bacterium divides, the plasmid also needs to divide and go with the daughter cells. If a plasmid cannot replicate in bacteria, then it will be lost.

9 Plasmid elements Antibiotic resistance: This allows for the presence of the plasmid to be selectively maintained in a given strain of bacteria Lab bacterial strains are sensitive to antibiotics. When grown on plates with antibiotics, they die. The presence of a plasmid with the antibiotics resistance gene allows these lab strains to grow on plates with the antibiotic. You are therefore selecting for bacterial colonies with the Plasmid

10 Plasmid elements Unique restriction sites: For cloning the plasmid needs too be linearized. Most cloning vectors have unique restriction sites. If the plasmid contains more than one site for a given restriction enzyme, this results in fragmentation of the plasmid

11 pUC18 pUC18 is a commonly used plasmid: pUC= plasmid University of California Plasmidrepliconcopy No pBR322pMB115 pUC18pMB1500 pACYCp15A10 pSC101pSC1015

12 Why are plasmids important? Most genes are present as two copies in the entire genome. Plasmids allow us to obtain 1000’s of copies of a gene in a pure form

Cloning The genomic DNA fragments is mixed with a plasmid that has been linearized at a single EcoRI site (say pUC18) Both the plasmid and genomic DNA have been cut with EcoRI, they have complementary sticky ends | G A A T T C C T T A A G |

14 Recombinant plasmid This process where foreign genomic DNA is joined to plasmid DNA is called ligation It results in recombinant plasmid (foreign DNA+plasmid) Each plasmid has one foreign EcoRI fragment Each foreign fragment is still present as only one copy! This is not useful.

15 Transformation The entire collection of these plasmids bearing genomic DNA inserts is called a Genomic Library! These plasmids are added back into bacteria by a process called transformation The bacteria are selected for the presence of the Plasmid by growth on media containing antibiotics Each colony of E. coli will harbor one plasmid with one piece of genomic DNA

Isolate the plasmid To isolate the gene C fragment, we grow up a large population of E. coli containing the plasmid with the gene C insert. A simple procedure allows us to isolate the plasmid (which is smaller than Chromosomal DNA) Once we have purified the plasmid we have 1000’s of copies of Gene C in a plasmid We can take the plasmid and cut it with EcoRI. When the digest is run on an agarose gel, we get two bands- one corresponding to the plasmid and one to the insert. The DNA present in the band corresponding to the insert can be isolated from the gel PURE GENE C!!!!!

17 Genomic clone libraries SpeciesGenome sizeaverage #plasmids insert size E. Coli5000kb16 kb1300 Drosophila150,000kb16 kb46,000 Human3000,000kb16 kb>100,000 The larger the genome, the more difficult the task of isolating a plasmid with a given gene At present, genomic DNA libraries exist for a large number of organisms including Yeast, C.elegans, Drosophila, Zebrafish, Xenopus, Chickens, Mouse, Humans etc

18 cDNA Often we have RNA rather than DNA as the starting material For instance in the case of the human hemoglobin gene, we started with globin mRNA RNA is difficult to work with. In contrast to DNA, RNA breaks down and degrades very easily. There are no restriction enzymes that cut RNA at specific sites. RNA cannot be cloned. It cannot be inserted into a plasmid and amplified since all Plasmids are DNA. The enzyme reverse transcriptase has proven very useful to molecular biologists. This enzyme catalyzes the synthesis of DNA from a RNA template. It is normally found in a large class of viruses. The genome of these viruses is RNA!! These viruses are called retroviruses.They infect eukaryotic cells and use these cells to grow/replicate Retroviruses carry an RNA genome. Interestingly they will integrate into the DNA of the host. For RNA to integrate into DNA, first the RNA has to be converted to DNA Remember the central dogma of molecular biology Information flows from DNA to RNA to protein! DNA---->RNA---->protein Reverse Transcriptase reverses this dogma (partially)

19 cDNA synthesis Protein coat RNA genome Reverse transcriptase

20 cDNA/splicing So from globin mRNA, a complementary DNA molecule can be created using reverse Transcriptase. This complementary DNA is called cDNA. This DNA can now be inserted into a plasmid and cloned. What is the relationship between a cDNA clone and a genomic clone? Splicing In eukaryotes, the coding sequences are interrupted by introns Gene 7700 nt Ovalbumin Splicing mRNA 1872 nt Primary transcript

21 Genomic clones represent the organization of the DNA in the nucleus! cDNA clones represents the organization of mRNA sequences after the gene has been transcribed, processed and exported to the cytoplasm. cDNA clones contain the sequence of nucleotides that code for the mRNA--protein! cDNA clones do not contain the sequence of the promoter of the gene or the intron. The starting material for cDNA clones is different from material used to make genomic clones Genomic clonecDNA clone SourceNucleiicytoplasmic RNA (any cell)(specific cell type) UseStudies on geneStudies directed organization &towards coding regions structure

22 Southern blotting Rapid method of identifying specific DNA fragments in a large mixture of fragments Marker EcoRI Uncut How do you determine which band corresponds to insert and which to the plasmid

23 A probe is used to identify genomic DNA? Marker EcoRI Uncut MarkerEcoRIUncut

24 Southern blotting with a probe The probe AAAAAAA will bind the single stranded DNA that has a complementary sequence. It will specifically hybridize with the insert (genomic DNA) A probe with this specific sequence is generated and made radioactive Incubate the filter with the radio- labeled probe A specific probe enabled us to identify a DNA fragment that corresponds to a specific gene of interest. TTTTTTT AAAAAA A

25 PROBES Probes are obtained in a number of ways RNA as a source The probe for hemoglobin can be obtained from mRNA of immature red blood cells. The major transcript of these cells is from the hemoglobin gene. So isolating RNA from these cells, we can obtain a relatively pure probe for the hemoglobin gene Protein If you have a purified protein, the amino acid sequence can be determined. From the amino acid sequence, using the genetic code a corresponding DNA sequence can be synthesized and this small DNA piece can be used as a probe Homology Probes from conserved genes-Many genes are conserved from one species to another Chimpanzee and human DNA are 97% identical. If you know the sequence of a gene in chimps, then you will be able to know the sequence for the gene in humans! The histone genes are highly conserved across phyla. Histone proteins have three Amino acid differences between humans and peas Histone genes have been isolated in yeast, they can serve as probes for screening a Human genomic library- cloning by phone The computer databases

26 What about a genome? What if Gene C was in a large genome. Could we identify the fragment by Southern blotting EcoRI 1kb2kb3kb4kb5kb4.5kb0.5kb GeneCGeneXGeneAGeneR

27 GeneC ProbeA ProbeB ProbeC EEEE 1kb2kb4kb3kb E

28 Gene EEEE EEEE

29 You can build a genomic restriction map If we digest the DNA with HindIII instead of EcoRI what will happen? GeneC EEEE HHHH

30 Rapid analysis of hemoglobin gene Exon1 MstII Exon2 0.2kb1.1kb 0.3kb deletion 

31 Northern blot This is a rapid method that allows you to determine the cell type in which a specific gene is active and being transcribed. Presence of RNA is a reflection of gene activity

32 Northern blot Method is analogous to Southern blots Instead of DNA as the starting material, you use RNA. You take cells, break them open, isolate the RNA and run the RNA on a gel Transfer RNA to membrane and use probe for gene of interest. The RNA can be from specific tissues or cell types Presence of RNA is a reflection of gene activity

33 Microarrays These are reverse northern blots. Allows us to examine gene expression of all of the genes in the genome! Each spot is DNA for one defined gene. Each gene DNA is spotted in a grid. They cover the entire genome. Make total RNA from normal and mutant cell, Label each total RNA differently Wt=red Mut=green Add labeled RNA from normal and mutant cells to array and let hybridize Measure label and determine change Ratio of WT/mut

34 Use of microarrays To measure changes in transcription of genes during drug treatment To identify deletions in DNA A microarray works by exploiting the ability of a given mRNA molecule to bind specifically to, or hybridize to, the DNA template from which it originated.

35 Animal cloning Animal clones are genetically identical. Natural clones occur in the form of identical twins but it is also possible to produce artificial clones by nuclear transfer. The nucleus is removed from a somatic (body) cell and placed in an egg whose own nucleus has been removed. The egg is then implanted in a surrogate mother and develops to term. Key principles * Differentiated animal cells are unable to develop into complete animals *The nuclei of most differentiated cells retain all the necessary genetic information. * Transfer such a nucleus into an egg whose own nucleus has been removed. * Transfer to the environment of the egg reprograms the nucleus (makes it forget its history) and allows the full development of a viable animal that is genetically identical to the donor of the somatic cell. * Until 1997, cloning in mammals was only possible using nuclei obtained from very early embryos. A breakthrough was made when cloning was achieved using nuclei from adult cells. * Recent research suggests that animals produced by cloning from adult cells may age prematurely, but further investigation is necessary. How does it work? Nuclear transfer is carried out by fusing the donor somatic cell to an egg whose own nucleus has been removed. Fusion is achieved in a culture dish by applying an electric current. The change in electrical potential also mimics the normal events of fertilisation and initiates development. A key aspect in the success of nuclear transfer is synchronisation of the cell cycles between the donor nucleus and the egg. Before fertilisation, the egg's nucleus is quite inactive. The nucleus of the donor cell must also be made inactive otherwise it will not be reprogrammed and development will fail. Inactivation is achieved by culturing the cell but starving it of essential nutrients. The cell stops dividing and enters a quiescent state compatible with nuclear transfer. How is it used? Animal cloning has the potential to overcome the limitations of the normal breeding cycle. In the future, it may be used to produce elite herds by cloning the superior animals, or to rapidly produce herds of transgenic or otherwise modified animals. Transgenic farm animals make useful bioreactors, producing valuable proteins in their milk. Another application is the use of genetically-modified pigs as a source of organs suitable for transfer to humans (xenotransplantation).