Homologous recombination and site specific recombination are two types of genetic recombination. Homologous recombination requires regions of high sequence similarity between two DNA strands and can result in the reciprocal exchange of DNA segments through the formation of Holliday junctions. Site specific recombination only requires short, specific recognition sites and is used by bacteria to integrate phage DNA. The Cre-Lox system is an example of site specific recombination that uses the Cre recombinase enzyme to catalyze recombination between LoxP sites, allowing for gene deletion, insertion and inversion.
Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica.- Source: Wikipedia
Introduction
Cre-lox recombination
Cre-lox system- Cre recombinase , loxP site
FLP-FRT recombination
FLP-FRT system- FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis , cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Exchange of genes between two DNA molecules to form new combinations of genes on a chromosome
contributes to a population’s genetic diversity (source of variation in evolution)
Recombination is more likely than mutation to be beneficial
Less likely destroy a gene's function
May bring together combinations of genes
Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica.- Source: Wikipedia
Introduction
Cre-lox recombination
Cre-lox system- Cre recombinase , loxP site
FLP-FRT recombination
FLP-FRT system- FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis , cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Exchange of genes between two DNA molecules to form new combinations of genes on a chromosome
contributes to a population’s genetic diversity (source of variation in evolution)
Recombination is more likely than mutation to be beneficial
Less likely destroy a gene's function
May bring together combinations of genes
Recombination in repair n damage of DNA.pptxANAKHA JACOB
• Maintaining a low mutation rate is essential for cell viability and health. It is estimated that both in prokaryotic and eukaryotic cells, DNA is replicated with very high fidelity with one wrong nucleotide incorporated once per 108–1010 nucleotides polymerized. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and post-replicative DNA repair systems.
• Mutations can occur due to errors in DNA replication as well as due to certain damages to the DNA. Errors in replication are corrected to a great extent by proofreading mechanisms. Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing many accidental lesions that occur continually. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair.• Maintaining a low mutation rate is essential for cell viability and health. It is estimated that both in prokaryotic and eukaryotic cells, DNA is replicated with very high fidelity with one wrong nucleotide incorporated once per 108–1010 nucleotides polymerized. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and post-replicative DNA repair systems.
• Mutations can occur due to errors in DNA replication as well as due to certain damages to the DNA. Errors in replication are corrected to a great extent by proofreading mechanisms. Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing many accidental lesions that occur continually. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair.• Maintaining a low mutation rate is essential for cell viability and health. It is estimated that both in prokaryotic and eukaryotic cells, DNA is replicated with very high fidelity with one wrong nucleotide incorporated once per 108–1010 nucleotides polymerized. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and post-replicative DNA repair systems.
• Mutations can occur due to errors in DNA replication as well as due to certain damages to the DNA. Errors in replication are corrected to a great extent by proofreading mechanisms. Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing many accidental lesions that occur continually. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair.
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritance.
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Homologous recombination and Site specific recombination
1. Homologous recombination and Site specific recombination
Presented ,
Mr-Pradeep D Devkate
M.Sc Microbiology, B.Ed, MH-SET, 2-GATE(Biochemistry, Zoology,
Botany)
2. Genome changes
Mutation Recombination
Sudden change in DNA Sequence
Mutagenic agents- Physical, chemical, Biological
Repair system work - Remove the Damage
and add the correct Nucleotides
Genomic Information is Stable.
Minimum changes due to Mutation
Rearrangement of DNA Molecules that involves the
Breakage and reunion of DNA
Minimum Homology required sharing information
between two strands
Due to recombination progeny differ from his parents
3. Recombination:
The process of exchange of DNA segments is known as Recombination
1) Homologous recombination
Similar or identical strands.
Results in the exchange of DNA segments.
Single stranded break = one holliday junction
DS break =two holliday junction
2) Site specific recombination –
Only a small site is similar in two DNA
Smaller DNA is integrated in to larger DNA
1)single strand break = tyrosine recombinase
2)double stranded break =serine recombinase
Between Two DNA Molecules that have only short
regions of homology Site specific recombination
Bacterial DNA phage
Exchange of homologous segments between two
homologous DNA molecules = H.R.
4. Transposons – Not required Homology
• Illegitimate Recombination--- Homology Independent
• Sequence of DNA inserted into Another regions without relying
sequence homology
• Self DNA Code Transposase Enzyme…. Cut to same DNA seq. and paste
to another site,,,,.
5. Homologous recombination:
It also involved in DNA repair.
Homologous recombination / general recombination
Also occur in all organisms but first discovered in bacterial system
Prokaryotic= conjugation, transduction, Transformation
Eukaryotic= In meiotic phase
Crossing over : reciprocal exchange of DNA segments
6. Holliday model for H. R. / heteroduplex model
Recombination between two homologous DS DNA molecules those
with identical or nearly identical sequences.
Ligation strand produces a crossed strand intermediate called as
Holliday junction
Junction moves / Branch migration exchange of longer segments of
DNA
Separation or Resolution of Holliday junction Cleavage across the
Branch Point
7. Step 1= Pairing of homologous DNA-
Step 2 = single strand break-( by Rec BCD)
Repair of UV Damage(RUV Protein)
9. Step 5- resolution (Ruv C)
Vertical cut = all recombinant
Horizontal cut= all parental
10.
11. 2) Site specific recombination or
conserved site specific recombination (CSSR)
• In Site specific recombination few nucleotide are similar between two
DNA .
• A specific site which are present called LOX-P Site.
att-B att-P
Integrase
att-L=BOP
att-R= POB
Sequence B And P are common in Both Phage and Bacteria is= O
After crossing over this attachment side
become hybrid ,,, and now they are known as
attachment-L and att-R ..
12. att-B att-P
Integrase
att-L=BOP
att-R= POB
o Lambda phage ds-DNA incorporated into Bacterial DNA called prophage.
o O is identical centre region of both= Short Homologous Region
o Tyrosine recombinase Break phosphodiester Bond and covalently attaches to 3’-
Phosphate group
13. Cre-Lox System
Cre = Causes Recombination (Recombinase Enzyme) – Tyrosine Recombinase
Lox P : Locus of X-gene Over P
Cre-Recombinase Recognizes 34 bp site in DNA called Lox-P site.
Cre-Recombinase catalysis Recombination between two Lox-P site.
It is a site specific recombination technology which is used to carry out
deletion, insertion , and translocation and inversion
it can used in eukaryotic and prokaryotic
14. Case 1- two Lox-P site are present in two
different DNA
• Result = Reciprocal translocation
15. Case-2 Two Lox-P site are present in same
DNA and Same Direction
• Deletion gene
16. Case-3 Two Lox – P-sites in same DNA and in
opposite direction
17.
18. • Lox-P site –same direction—Deletion of gene
• Lox –P site opposite site ---- Inversion of Gene
• Lox-P site on different chromosome--- Reciprocal Translocation
19. Two strands are 99 % similar , in this strands
which type of recombination is possible ???
20. Proteins Involved in Homologous Recombination in Prokaryotic
Rec B,C,D – processing of ds DNA break.(Helicase , Nuclease Activity)Exonuclease-V
step-2= Rec-D 5’3’ exonuclease (ATP Dep)
Rec-B 3’5’ Exonuclease (ATP Dep)
Activity of enzyme is altered when it interact with Chi sequence(5’GCTGGTGG3’) in E.Coli.
RecA first binds cooperatively to the invading single strand, help to pairing of homologous strands.
Rec-C Recognized Chi Sequence
Rec-C Signal to Rec-D to stop Unwinding of DNA
RUV-A,B – Branch Migration steps-10-20 bp/sec
RUV-A specific DNA Binding Protein , Recognize and Bind to Holliday junction and Recruits
RUV-B Protein
RUV-B ATPase Activity provide energy for Branch Migration
RUV-C – Resolution ( Holiday Junction)
cleavage Resolves of the holliday junction
Unwinding
1.Rec A,B,C= Recombination Protein A,B,C.. 2.RuvABC (Recombination UV)