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Gene engineering & Gene expression in vitro

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  1. Gene engineering & Gene expression in vitro

  2. Genentech Founders • 1973,Stanley Cohen and Herbert Boyer DNA recombination: pSC101 RSF1010 pSC109 E coli C600

  3. DNA recombination concept: DNA molecule from different scources covalently ligated with phosphodieaster bond to produce a noval DNA molecule

  4. Basic concepts • genetic engineering are terms that apply to the direct manipulation of an organism's genes. It uses the techniques of molecular cloning and transformation to alter the structure and characteristics of genes directly. Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. Cloning is frequently employed to amplify DNA fragments containing genes, but it can be used to amplify any DNA sequence such as promoters, non-coding sequences, chemically synthesised oligonucleotides and randomly fragmented DNA.Its core technique is DNA recombination clone In genetics, an exact replica of all or part of a macromolecule (e.g. DNA) In cell biology, a group of identical cells naturally derived from a common mother cell; of significance in vertebrate physiology, and concepts related to immunology and cancer biology

  5. Section 1 Tool enzymes • DNA recombination technique is hghly dependent on tool enzymes and various vectors • Tool enzymes: incision, synthesis, ligation and modification

  6. 一、restriction enzyme • an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. • Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. • these two processes form the restriction modification system.

  7. 1. Classification of restriction enzyme • Typical type II restriction enzymes differ from type I restriction enzymes in several ways. They are composed of only one subunit, their recognition sites are usually undivided and palindromic and 4-8 nucleotides in length, they recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity – they usually require only Mg2+ as a cofactor.In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily nomenclature was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes.[15] These subgroups are defined using a letter suffix.Type II of restriction enzymes are widely used

  8. 2. Nomenclature of restriction enzyme Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain.

  9. 3. Characterics of type II restriction enzyme • Recognization site: undivided and palindromic and 4-8 nt in length • Rsa I EcoR I Not I • GTAC GAATTC GCGGCCGC • CATG CTTAAG CGCCGGCG • 6nt palindromic sequence is widely used the frequency of cutting site: 46=4096 bp • Special recognization site: BstE II EcoR II • GGTNACC CC GG A T

  10. Cutting modes: Sticky end:5,overhang 3,overhang Blunt end • DNA molecule with same end can be recognized and ligated。

  11. isoschizomer and isoaudamer • Isoschizomers are pairs of restriction enzymes specific to the same recognition sequence. BamH I and BstI--G↓GATCC • Xho I and PaeR7 -- C↓TCGAG • isoaudamer An enzyme that recognizes slightly different sequence, but produces the same ends BamH ISau3A I GGATCCNGATCN CCTAGG NCTAGN

  12. 4. Application of restriction enzyme • Essential tool for DNA recombination • DNA cutting • Mapping the physical map of genome • Detection of DNA mutation (RFLP)。 • Reaction for restriction enzyme: • Mg2+ • pH,neutral condition • Ionic strength with different concentration of Na+ or Ka+

  13. Restriction digestion of lamda bateriaophage DNA

  14. 二、modifcation enzyme • (1) DNA polymerase I:MW 109KD • 5,→3, polymerizarion • 3,→5,exonuclease activity • 5,→3,exonuclease activity • application: nickle translation,the second strand synthesis of cDNA, DNA 3’end labeling DNA pol I 5 3

  15. Klenow • Klenow fragment(76KD): • 5,→3, polymerization and 3,→5,exonuclease activity • Blunting the 3’end; • Labeling the 3’end • Synthesis of the second strand of cDNA • DNA sequencing • Taq DNA polymerase: • Cloned from Thermus aquaticus • No proof reading activity and the optimal temperature is 72℃ • Used in PCR 蛋白酶

  16. 2、reverse transcriptase • Reverse transcriptase,RNA-dependent DNA polymerase, is a DNA polymeraseenzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of this • types:AMVand MMLV • activity: • reverse transcriptase • RNase H DNA pol • RNase H deleted • Primer is needed for RT。

  17. 3、DNA ligase • DNA ligase is a special type of ligase that can link together two DNA strands that have double-strand break (a break in both complementary strands of DNA). The alternative, a single-strand break, is fixed by a different type of DNA ligase using the complementary DNA as a template but still requires DNA ligase to create the final phosphodieaster bond to fully repair the DNA • Ligase mechanism The mechanism of DNA ligase is to form two covalent phosphodieaster bond between 3’ hydroxyl end of one nucleotide with the 5’ phosphate end of another. ATP is required for the ligase reaction. • Ligase works both with blunt and sticky ends of DNA. T4 DNA Ligase: • Ecoli DNA ligase

  18. 4、alkaline phosphatase • Alkaline phosphatase is a hydrolaseenzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. • Calf intestinal alkaline phosphatase (CIAP): Application: prevent vector self-ligation • 5’end labeling • enzyme-linked developing

  19. 5、Terminal deoxynucleotidyl transferase(TdT) • TdT catalyses the addition of nucleotides to the 3' terminus of a DNA molecule. Unlike most DNA polymerases it does not require a template. The preferred substrate of this enzyme is a 3'-overhang, but it can also add nucleotides to blunt or recessed 3' ends. • Highest activity with 3’end overhang • Labeling of 3’end Adding homo-polytail cpnstructing artifical sticky end

  20. Section 2 vector • Vector:vehicle used to transfer genetic material to a target cell • Characterics of vectors: • orgin • Selection marker • Multiple cloning site • Capacity

  21. Vectors which host is Ecoli • plasmid • λbacteriophage • cosmid • M13phage

  22. 一、plasmid • A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae

  23. Natural plasmid classification • Fertility-F-plasmids, which contain tra-genes. They are capable of conjugation (transfer of genetic material between bacteria which are touching). • Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. Historically known as R-factors, before the nature of plasmids was understood. • Col-plasmids, which contain genes that code for (determine the production of) bacteriocins, proteins that can kill other bacteria. Features of Plasmid used in molecular biology • Relativel small molecula weight • Drug resistance gene • Multiple cloning site • Orgin site for replication

  24. Application of plasmids cloning Sequencing In vitro transcription Gene expression

  25. Main types of plasmids in molecular biology • pBR322 • most commonly used E. colicloningvectors. • the first artificial plasmid. Created in 1977, it was named eponymously after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez. • 4363 base pairs in length and contains a replicon region (source plasmid pMB1), the ampR gene, and the tetR gene,(source plasmid pSC101). • The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites for HindIII and within the promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene. The origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1)[2]. The ori encodes two RNAs (RNAI and RNAII) and one protein (called Rom or Rop).,

  26. pUC18 and pUC19: • High copies in cell • Containing LacZ to faciliate the recombinant selection • MCS containing more enzyme recognization site • pUC118/119: • Repilcation origin based on pUC18/19

  27. 二、λbacteriophage • The virus particle consists of a head and a tail that can have tail fibres. The head contains 48,490 base pairs of double-stranded, linear DNA flanked by 12-base-pair, single-stranded segments that make up the two strands of the cos site. In its circular form in the host cytoplasm, the phage genome therefore is 48,502 base pairs in length. The prophage exists as a linear section of DNA inserted into the host chromosome • Lytic or lysogenic

  28. Charterictics of λphage • Big genome,packing into virus particle to infect cell • Cos site is needed for packaging, recombinant size ranging between 75%-105%of genome • 40%of genome can be replaced. The longest insert is 23kb

  29. Types of λphage • Two types : • Insetion vector:one restricition enzyme recognization site。 • theoritical capacity:0-13.5 kb • Replacement vector:two RE recognization site • theoritical capacity:9-22.5 kb

  30. Types of λphage DNA • Charon 40:replacement, insert 9.2-24.2 kb • EMBL 3:replacement, insert 7-22 kb。 • Spi selection • λgt 10:insertion ,insert 0-7.6 kb • λgt 11:insertion,insert 0-7.2 kb • LacZgene selection • λDASH:replacement,insert 9-22 kb • Spiselection,T3 and T7 promoter • λZAP:insertion,insert less than10 kb • ColE1 replicon,convert to plasmid

  31. 三、cosmid • A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid (often used as a cloning vector) that contains cos sequences, DNA sequences originally from the lambda phage. Cosmids can be used to build genomic library. Insert 40-45 kb

  32. Features of cosmid • advantages • Ampr or Tetr • Cos site,in vitro packaging • One or more RE recognization site • High capacity,especially for genomic library construct。 • Non-recombinant cosmid is small, can not be in vitro packaged, easily for selection • disadvantages The length of insert affect the amplification efficiency

  33. 四、Filamentous phage vector • Phage particle is filamentous, circular single-strand DNA genome, the length is 6.5kb M13 , f1,fd • M13 only infect the Ecoli with F factor • M13mp18/M13mp19: • Containing LacZ and MCS • Limited packing capacity,used in sequencing。

  34. Other vectors YAC:capacity 0.5-2MB

  35. BAC:capacity 0.1-0.4MB

  36. expressing vector • An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery. • Expression vector is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA • Host cell:prokaryote and eukaryote

  37. (一)expression vector in Ecoli promoter SDsequence terminator Expression element

  38. Commonly used prokaryotic promoter • Tac promoter: • heterogenous promoter with Trp (-35), artifical synthesised -10 regionsandLac operator. Containing LacI • very strong promoter,can be induced byIPTG • T7phage promoter:highly specific promoter,only expressed in T7 RNA polymerase-containing host cell. • Lac operator in the downstream of T7 promoter, and can be induced by IPTG • pL promoter of λ:controlled by temperature-sensitive inhibitors(cIts857),repress transcription at low temprature and ddoes not work at high temparature

  39. S D sequence • Located between transcription starting site and initiation • The distance between SD sequence and initiation codon is very important to transcription activiety

  40. transcription termination sequence • length:to 800bp • characterics:strong termination for RNA ploymerase, A/T rich and G/C rich Palindromic structure

  41. (二)expression vector in mammalian • Basic element needed : ori, resistance gene, MCS • Element needed for expression: • Promoter and enhancer:from virus, strong activity • Poly A tailing:AAUAAA • Splicing signal:provided by insert • Selection marker:tk and neor

  42. Section 3 Process of gene cloning

  43. 一、preparation of target DNA first and very important step for cloning Strategy for gene cloning positional clonging: RE digestion directional cloning Sequence-based cloning: PCR chemical synthesis capture from library Function-based cloning:immuno selection

  44. (一)preparation of genomic DNA • genomic library: a population of host bacteria, each of which carries a DNA molecule that was inserted into a cloning vector, such that the collection of cloned DNA molecule represents the entire genome of the source organism. This term also represents the collection of all of the vector molecules, each carrying a piece of the chrosomal DNA of the organism, prior to the insertion of these molecules into the host cells. • Vectors: plasmid, λphage, YAC • shotgun • Use: Sequencing for whole genome

  45. (二)preparation of cDNA • A cDNA library is a collection of cloned cDNA fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. • cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism.

  46. Full length cDNA synthesis • SMART: Switch mechanism at RNA terminal

  47. Characterics of cDNA • Expressed library • Lack of intron, and can be expressed in prokaryote • tissue specific cDNA libraries • Low abundant mRNA is difficult to get from cDNA library