Thông tin tài liệu
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Enzymes needed:
1.Restriction enzymes: cuts the DNA at specific
sequences to generate a set of fragments
2. DNA ligase: inserts DNA restriction fragments
into replicating DNA molecules to produce
recombinant DNA
Lambda vectors
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677###7&7*00708
EcoRI
EcoRI EcoRI
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limitations
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Vector types:
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Vector Insert size
(kb)
Plasmid <10 kb
Bacteriophage
λ
9-15 kb
Cosmids 33-50 kb
YACS 100-1000 kb
What determines choice of vector?
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# C9
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-Central 1/3 is the “stuffer” fragment.
-Segments of the lambda DNA are removed and a stuffer
fragment is put in, this keeps the vector at a correct size
•
[...]... responsible for DNA recombination and integration of the prophage, as well as cII and cIII -Q is an antiterminator similar to N, but only permits extended transcription from PR -Two Termination sites- One between N and CIII and other between cro and CII • http://www-micro.msb.le.ac.uk/224/Phages.html #Lambda Life cycle of lambda 1 2 3 4 5 6 7 8 9 Virus enters cell PL and PR gets activated PL transcribes... can now make cIII and cII, replication proteins (O and P) and Q) There are also termination sites next to Q protein Q protein will allow transcription past this site If Cro protein blocks production of cI (goes lytic) If cII and cIII activates transcription to make cI (goes lysogenic) cI blocks PL and PR (stops transcription) by binding to OL and OR How do cells leave lysogeny cycle and go to lytic... OL, preventing transcription of cro and N, but allowing transcription of OL, and the other genes in the left hand end -cII and cIII encode activator proteins which bind to the genome -Cro (66 aa) protein which binds to OR and OL, blocking binding of the repressor to this site to prevent lysogeny -N codes an antiterminator protein and allows transcription from PL and PR It also allows RNA polymerase... the cell to adapt to and survive in altered environmental conditions RecA cleaves the cI repressor protein Which proteins determine the cycle? • Lysogenic cycle: cI proteins predominate • Lytic cycle: cro proteins predominate DNA lambda replication • Initation of replication at the lambda origin requires “activation” by transcription starting from PR • DNA replication is between O and P gene proteins... AT rich region) • • • • • • • DNA Replication, W.H Freeman and Co (1992) Kornberg,A • O protein binds to lambda origin causing a structural change in the origin P protein interacts with O protein Lambda proteins O and P form a complex with DnaB at the lambda origin (complex is inactive) This forms a spherical structure called an “O-Some” (~100bp of DNA) P protein (lambda s) brings dnaB to the origin... 3rd Edition, Garland Publishing, Inc 1983 • http://wwwmicro.msb.le.ac.uk/224/Phages.html #Lambda -PL ( promoter) for transcription for the left side of λ with N and cIII -PR (promoter) for right, including cro, cII and the genes encoding the structural proteins -OL and OR is short non-coding region of genome, they control the promoters -cI (repressor) protein of 236 a.a which binds to OR and OL, preventing... Shock proteins (dnaK, dnaJ and grpE gene) dissociate the oriλ O.P.dnaB complex to liberate dnaB dnaB initiates unwinding of duplex Primase starts chain initations and polII starts elongation −λ circles multiply by DNA Replication, W.H Freeman and Co (1992) Kornberg,A theta form(θ) and continues for 5-15 minutes after infection -Rolling circles predominates after 15 minutes and produce linear concatemers... Prentice Hall, Madigan, Martinko, Parker Recombinant DNA: A short course, W.H Freeman and Co.(1983) Watson, Tooze, Kurtz http://www-micro.msb.le.ac.uk/224/Phages.html #Lambda DNA Replication, W.H Freeman and Co (1992) Kornberg,A Genes VII, Oxford Unine Press (2000), Lewin Benjamin http://www.biocan.com/pdf/FAQ%20TrueBlue%2 0Vectors. pdf http://www.cc.ndsu.nodak.edu/instruct/mcclean/plsc731/cloning/cloning1...• Origin of replication is a DNA segment recognized by the cellular DNAreplication enzymes Without replication origin, DNA cannot be replicated in the cell http://www.uic.edu/classes/phar/phar331 /lecture6/ • Selective marker is required for maintenance of... http://www.uic.edu/classes/phar/phar331/lecture6 http://www.gmu.edu/departments/biology/385-Ch04c-rDNA/ steps in cloning with λ : – Isolate vector DNA and gene of interest – Cut both with restriction enzyme(EcoRI) – Connect two fragments of foreign DNA with DNA ligase (recombinant DNA) – Package DNA by adding cell extracts containing head and tail proteins – Transfer recombinant molecules into host cell (transform) – Grow/select transformants: . fragments
into replicating DNA molecules to produce
recombinant DNA
Lambda vectors
•
0(
•
(5
•
677###7&7*00708
EcoRI
. $&D
-Central 1/3 is the “stuffer” fragment.
-Segments of the lambda DNA are removed and a stuffer
fragment is put in, this keeps the vector at a correct
Ngày đăng: 13/03/2014, 18:46
Xem thêm: Lambda vectors and their replication, Lambda vectors and their replication, -Central 1/3 is the “stuffer” fragment. -Segments of the lambda DNA are removed and a stuffer fragment is put in, this keeps the vector at a correct size, Molecular Biology of the Cell, 3rd Edition, Garland Publishing, Inc. 1983
