CYBRIDS

 Cybrids- It is also called as cytoplasmic hybrid.  It consist of 2 protoplast one is donor and other is recipient the nuclear content is eliminated forming a normal heterokaryon while its plastome(cytoplasmic genome) or chondriome (mitochondrial genome )content are merged with the recipient. 

• Cybrids are cells  containing nuclear genome from only one parent but cytoplasm from both the parental species. The process of protoplast fusion resulting in the development of cybrid is known as cybridization.

• It possess heterozygosity of extrachromosomal genes. It may be single cell or a complete growing plant called cybrid plants.

Applications-

1.Way for those species witch don’t perform to sexual reproduction with each other, so this way provided to facilitate to make a desire species with combination of both species.
2.Production of wide range of genetic variations. 
3.Useful for sterile plants, mitochondrial gene also combined with chloroplast genes of another species.


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S1 nuclease mapping

S1 nuclease mapping -
    
S1 nuclease is an endonuclease which is  specific for hydrolyzing single-stranded RNA or DNA molecules. (zinc-requiring ) It is more active on DNA then RNA. The protein is thermostable and resistant to several denaturing agents, such as urea, SDS, and formamide . 
m-RNA  is hybridized to ss DNA( radiolabelled with a probe ) that overlaps the start of the target transcript . It  results in DNA-RNA hybrid. The hybrid molecule has single stranded extensions that are degraded by the single stranded specific S1 nuclease . The 3' end of DNA fragment has been removed by using electrophoresis .

                        Image source-http://biocadmin.otago.ac.nz/fmi/xsl/bioc2/learnbitslecture.xsl?-db=BIOC2web.fp7&-lay=Lectures&-recid=4841&-find=

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Melting temperature (Tm)

(Melting temp.-Tm)

The temp. at which half the strands of DNA are single stranded and half are double stranded. Half of the DNA are denatured . (Tm) It  is characteristics of Dna compostion ( GC content) .The higher the G+C content higher the Tm. 

                                               

                                                           

Image source-http://cbc.arizona.edu/classes/bioc461/Chapter5Part1Notes.htm

Image source-http://www.biology.arizona.edu/biomath/tutorials/linear/linearfunctionapplication/dnamelt.html

            

                      Tm directly proportional to G+C content 

Q.What is the maximal t_m for a DNA sample that is 500.0 bp long?

Tm is useful for monitoring your PCR reaction because it lets you distinguish between specific and unspecific amplification. Of course it is easy to see if you have dimer formation if you run your PCR products on a gel, but

with many samples it soon becomes tedious.

You can monitor DNA melting through UV light. The aromatic bases in DNA absorbs UV light (260nm). But when the two strands are hydrogen bonded with each other, the UV light is not absorbed readily. However, when they melt, the bases are exposed and the absorption goes really high. So if you shine UV light onto your sample and raise the temperature of your sample, you will see a sudden increase in absorbency when the DNA melts.

The Tm can be determined experimentally or calculated from simple formula -

   

    Tm=(4x [G+C]) + 2x[A+T]) 

Factirs that affect the Tm -
1.Concentration of DNA.
2.Concentration of ions in the solution ,most notably Mg2+ and K+.
3.DNA sequence
4.Length of Dna.

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