Restriction enzymes are molecular scissors which cut DNA at specific base sequences. E.g. HindIII and EcoR1 are restriction enzymes Hind III cuts DNA at the recognition sequence 5’-AAGCTT-3’ EcoR1 cuts DNA at the recognition sequence 5’- GAATTC-3’ Restriction Enzymes fig 14.2
Restriction Enzymes: (Fig 14-1) Many of the sites in DNA which restriction enzymes recognize are PALINDROMIC Palindromic: means that the sequence reads the same 5’-3’ in both directions
Genomic library Collection of DNA fragments that represent all the DNA in the genome Chromosome library All the DNA fragments in that specific chromosome cDNA library Produced using reverse transcriptase Makes DNA copies of mature mRNA
Types of PCR I RT PCR Reverses transcriptase PCR. DNA is amplified from mRNA. QRT PCR Quantitative Real Time PCR the amplified product is linked to a fluorescent reporter molecule, the fluorescence is measured at each cycle. This allows the amplification to be monitored to optimize the efficiency of amplification.
Types of PCR II Multiplex PCR two or more sets of primers are used in the same reaction mix. Nested PCR (also nested RT-PCR) There are two amplifications made: the: The first amplifying a large product which, in the second amplification is used as the template.
Gel Electrophoresis A method of separation pieces of DNA Horizontal gel (Agarose) Vertical (Polyacrylamide) Many variations on the methods exist
The DNA is negatively charged and migrates towards the positive pole The smallest fragments move the fastest through the gel and therefore move the furthest The largest fragments move the least By comparing the fragments in the sample with standards of known size the size of the sample fragments can be estimated
Links to virtual labs DNA extraction http://learn.genetics.utah.edu/units/ biotech/extraction/ Making a gel http://learn.genetics.utah.edu/units/ biotech/gel
Stem Cells are undifferentiated cells Type of Stem CellDevelopmental Potential Early EmbryonicTotipotent Blastocyst EmbryonicPluripotent FetalPluripotent UmbilicalMultipotent AdultMultipotent http://learn.genetics.utah.edu/units/stemcells