Molecular Genetics and Genetic Engineering GEN 301 Lecture 1 PDF

# Molecular Genetics and Genetic Engineering GEN 301 Lecture 1 ### Presented and edited by Dr. Mustafa Mahmoud, Ph.D. Faculty of Biotechnology October University for Modern Sciences and Arts (Fall 2024) ## Introduction ### Molecular Biology: - Field of science concerned with studying the molec...

# Molecular Genetics and Genetic Engineering GEN 301 Lecture 1 ### Presented and edited by Dr. Mustafa Mahmoud, Ph.D. Faculty of Biotechnology October University for Modern Sciences and Arts (Fall 2024) ## Introduction ### Molecular Biology: - Field of science concerned with studying the molecular nature of the gene and its mechanisms of gene replication, transcription, translation, etc... ### Molecular Genetics: - Molecular genetics relies heavily on genetic engineering (recombinant DNA technology), which can be used to modify organisms by adding foreign DNA, thereby forming transgenic organisms. ### Chronology: - 1973: created first genetically modified (GM) bacteria - 1974: created GM mice - 1982: first commercial development of genetically modified organisms [GMOs] (insulin-producing bacteria) - 1994: began to sell genetically modified food ## DNA recombination ### Restriction enzymes (restriction endonucleases): - enzymes, which are naturally produced by bacteria - cut **DNA** at a particular sequence of bases (recognition site): - 4-8 bp - Palindromic sequence | Enzyme | Recognition sequence | Cutting sites | Ends | | :-------- | :-------------------- | :---------------- | :-------- | | BamHI | 5'- GGATCC-3' | GGATCC / CCTAGG | sticky | | EcoRI | 5'- GAATTC-3′ | G'AATT C / CTTAAG | sticky | | Haelll | 5'- GGCC-3′ | GGCC / CCGG | blunt | | Hpal | 5'- GTTAAC-3′ | GTTAAC / CAATTG | blunt | | Pstl | 5'- CTGCAG-3' | CTGCAG / GACGTC | sticky | | Sau3A | 5'- GATC-3′ | GATC / CTAG | sticky | | Smal | 5'- CCCGGG-3' | CCCGGG / GGGCCC | blunt | | Sstl | 5'- GAGCTC-3' | GAGCT C / CTCGAG | sticky | | Xmal | 5'- CCCGGG-3' | CCCGGG / GGGCCC | sticky | #### Types: - **Type I**: cuts **DNA** randomly (upstream or downstream) the recognition site by 1000-5000 - endonuclease and methylase activities - **Type II**: Cuts **DNA** within or near recognition sequence - endonuclease activity - **Type III**: Cuts **DNA** 20-30 bp downstream recognition site - endonuclease and methylase activities - **Type IV**: Recognizes modified (methylated) **DNA** - endonuclease activity - **Type V (Artificial restriction enzymes)** - Generated by fusing a **DNA** binding domain to a nuclease domain. - They can cut **DNA** of variable length, provided that a suitable guide **RNA** is provided. - ex. CRISPR-Cas system, TALENS, Zinc finger nucleases #### Type II Subgroups: | Subgroup | Mechanism of digestion | | :-------- | :------------------------------------------------------------------------------------ | | Type IIP | Cut at the recognition site | | Type IIS | Cut away from the site | | Type IIB | Require two recognition sites and cut on the outside | | Type IIE | Require two recognition sites, and one of the two sites acts as allosteric effector | | Type IIF | Require two sites and cut at both sites as a tetramer after bringing the two regions together by looping the **DNA** | | Type II | In contrast to most Type II REs that act as dimers Bcnl act as a monomer | #### Concepts: - **Neoschizomers**: Different REs recognizes the same sequence but cut it differently. - **Isoschizomers**: Different REs recognize the same sequence and cleave in the same location. - **Isocaudomer**: Different REs that recognize and cleave different sequences giving compatible or complementary sticky ends. - The ligation of these sticky ends produces new sequence that can’t be digested by any of them. ### Restriction Mapping - To construct a map showing the relative positions in the DNA molecule of the recognition sequences for a number of different enzymes. - Important to select the correct RE for the particular cutting. #### Example: *What would be the number and sizes of the fragments that you would expect to find if you cut the following **DNA** fragment with: i- Enzyme A, ii- Enzyme B, iii- Both enzymes together?* | | Enzyme A | Enzyme B | Both | | :-------- | :------- | :------- | :------ | | | 1000 bp | 1200 bp | 1000 | | | 2100 bp | 2500 bp | 200 | | | 1400 bp | 1300 bp | 1900 | | | 500 bp | | 600 | | | | | 800 | | | | | 500 | #### Example: *Linear **DNA** fragment was digested. The reaction along with the fragments obtained in single and double digest reactions, resulted in:* | | BamHI | EcoRI | BamHI + EcoRI | | :--------- | :------- | :------- | :------------- | | | 14 | 12 | 11 | | | 1 | 3 | 3 | | | | | 1 | *Using this information, construct a restriction map of the **DNA** fragment.* #### Example: *An isolated plasmid was digested using EcoRI and BamHl. The fragments obtained in single and double digest reactions were as following:* | | EcoRI | BamHI | EcoRI + BamHI | | :------ | :------ | :------ | :------------- | | | 20 kb | 6 kb | 6 kb | | | | 12 kb | 4 kb | | | | 2 kb | 8 kb | | | | | 2 kb | *Using this information, construct a restriction map of the plasmid.* ### Ligases: - Catalyzes the formation of phosphodiester bond - NB: - Cut both the insert and vector by Isoschizomer RE so they give sticky ends complementary to each other giving. - Blunt end ligation between insert and vector is an inefficient process. - Vector and the insert can ligate in only one orientation (i.e., directional) and the digested ends of the vector are incompatible for self-ligation. - Possible outcomes for ligation: - i- recombinant vector; ii- native vector; iii- insert / vector concatemers; iv- no ligation #### Types: - **Bacterial DNA ligase** - 1- E.Coli ligase - 2- Needs NAD+, NADP as energy source - 3- Efficiency of ligation is low so can’t join blunt end (except under conditions of macromolecular crowding) - **Bacteriophage Ligase** - 1- T4 DNA ligase (from T4) - 2- Needs **ATP** as source of energy - 3- Efficiency of ligation is high so can join both sticky and blunt ends ### Blunt end ligation with a DNA topoisomerase - efficient way for blunt end ligation - DNA topoisomerases have both nuclease and ligase activities. - topoisomerase cuts DNA at the sequence CCCTT, which is present just once in the plasmid - after cutting the plasmid, topoisomerase enzymes remain covalently bound to the resulting blunt ends - insert is produced by cutting with a restriction enzyme, and treated with alkaline phosphatase to remove terminal 5’ phosphates before mixing the insert with the vector - only one strand is ligated at each junction point and the nicks will be repaired by cellular enzymes after the recombinant molecules have been introduced into the host bacteria #### Example: - [Graphic depiction of Blunt TOPO Cloning] ### Cloning with Different End Types: - **Cloning with two sticky ends** - Sticky ends must be compatible - Cloning is directional - Insert-vector ligation is efficient - Vector self-ligation is low - Recognition sites of ligated restriction enzymes are intact - **Cloning with two different but compatible ends** - Sticky ends must be compatible - Cloning is directional - Insert-vector ligation is efficient - Vector self-ligation is low - Recognition sites of original restriction enzymes (e.g., Xhol and Sall) may be destroyed after ligation - **Cloning with one sticky end and one blunt end** - Directional cloning is maintained - Ligation of the blunt ends may be less efficient - **Cloning with blunt ends ** - Ends are compatible - End sequences are modified - Directional cloning is lost - Ligation may be less efficient - Vector self-ligation is high ### Conversion of blunt ends to sticky ends - Addition of linkers or adaptors or homopolymer tailing to blunt ends: #### Linkers: - Double-stranded-oligomers that contain a recognition sequence for a particular restriction enzyme is added to blunt ends followed by digestion with restriction endonucleases. #### Adaptors: - Short double-stranded sticky ended oligonucleotides added to blunt ends, and do not require further digestion with RE #### Homopolymer Tailing: - Enzyme terminal transferase (TdT) adds homopolymers of dA, dT, dG, or dC to 3' of **DNA** molecules (ex. dG tails on insert and dC tails on vector) #### NB. - TdT add from 2- to 15-mers - homopolymer tailing advantages over the other methods: - provide longer regions for annealing **DNAs** together: means that ligation need not be carried out in vitro, as the **DNA**-vector hybrid is stable enough to survive introduction into the host cell, where it is ligated in vivo - more specificity: as the vector and insert **DNAs** have different but complementary tails, there is little chance of self-annealing, and the generation of concatemers #### Restriction sites introduction into PCR products: - PCR primers are designed which recognize sequences flanking the target region - A short extra region of **DNA** encoding a restriction site is added to the 5' end of the primers - The 5' ends of the primers with the restriction site will be incorporated into the newly synthesized amplicons ## Thank you For ## Your attention