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Restriction endonucleases

-bacterial enzymes designed to destroy intruder DNA (phage) creating DNA fragments


modification-restriction systems
component and their role

-includes methylase and an endonuclease
-methylase causes methylation at a specific sequence, protecting the bacteria from the endonuclease
-endonuclease cleaves at specific sites of nonmethylated sequences


Eco R1
site that it recognizes

restriction endonuclease that recognizes GAATTC
-notice that the site is palindromic


REs and gel electrophoresis

-DNA fragmented by REs can be ran through a gel and seperated by size


size of the DNA fragments

cut roughly around every 4kb, therefor there are many genes per fragment


pulsed gel electrophoresis

used to seperate very large pieces of DNA, on the order of full chromosomes


Southern Blot

-used to identify DNa fragments under study
-use an RE to make DNA fragments
-run these fragments on a gel
-blot the gel on a membrane (nitrocellulose) that tightly binds DNA so it transfers
-during the transfer, the DNA is denatured, exposing the nitrogenous bases
-The fragment of interest is then identified by the hybridization (complementary base pairing) of a radioactive labeled DNA or RNA probe
-the probe will make a mark on X-ray film, this is called autoradiography


restriction map

shows were various restriction endonucleases will cut around aspecific region (ie target gene)


Identifying insertions and deletions

if you have a RE that cuts on either side of an insertion or deletion, the DNA fragment will change in size and run slower/faster on a gel. then visualized via southern blot


identifying point mutations

if the point mutation is within the restriction site then the RE will not longer cut at this site on the mutated DNA strand. therefore, when ran on a gel, the fragment will be longer than the wild type strand.
-this can be done for patients with sickle cell anemia. if the patient is heterozygote for the trait, the gel will show both the long (mutated) and short (wild type) band


restriction fragment length polymorphism
general concept
clinical significance

-there are many areas of the human gene the differ between every person, therefore every person wil have some different sized DNA fragments aftet being subjected to an RE
-however some fragments are novel and may contain a gene that if mutated could cause disease. if there is a change in this novel framgnet, you know that there was a mutation


Northern Blots

-used to detect RNA molecules
-RNA ran through a denaturing gel (keeping the RNa single strnaded)
-transferred to a membrane
-visualized using a radiolabeled DNA or RNA probe using autoradiography


Western Blot

-used to identify a specific protein
-a protein solution is ran through an SDS polyacrylamide gel. the SDS denatures the proteins and equalizes the charge on the proteins allowing them be seperated solely on size.
-after they are ran, they are transferred to a polymer sheet
-a specific radiolabeled antibody is added
this process relies on protein protein interaction whereas souther and northern are nucleic acid-nucleic acid


Electrophoretic mobility shift assay

used to determine if a certain protein associates with a certain segment of DNA
-radiolabelled DNA is mixed with the protein of choice then ran on a gel
-if the DNA doesnt run as far as the naked DNA then you can assume it has been slowed down by the binding of the protein


DNAse 1 footprinting

-allows one to determine the boundaries of a DNa-protein interaction
-first label the DNA strand at a single end
-ad the protein to the labelled DNA
-treat the sample lightly with DNAse
-regions of DNA protected by the protein will remain uncut
-therefore when you run all of your different sized DNA fragments on a gel, there will be a gap when the protein reside


polymerase chain reaction
three steps of the process in detail

-DNA amplification technique
-repetative cycling of three reactions: denaturation, annealing, and extension
-denaturation is the first step and is done by increasing the temperature so the DNA strands seperate (90C)
-Annealing consists of lowering the temp back down to below what is requier to denature, during this process, two oligonucleotide primers are annealed to their compliments
-these primers define the limit of DNA being amplified
-the longer the primers, the more specific the annealing (1/4) to the N, N=number of nucleotides
-extension is the last step and involves synthesis of a ned strand using taq polymerase which starts at the primers. dNTP's are required



-determine whether an RNA molecule of interest is present in the sample
-initially, reverse transcriptase is used to convert RNa into single stranded cDNA
-cDNA is the amplified using standard PCR methods


Single Strand Conformation Polymorphism

-can be used to identify mutations
-PCR product is chemically seperated into single strands (formamide) and ran on a gel.
-these strands will migrate according to their conformation
-if there is a mutation, that conformation will change and the strand will run differently


insertion of restriction or PCR fragments into self replicating vectors

-permit amplification and purification of fragments of DNA of interest
-Plasmids are removed from bacterial cells and cleaved using the same restriction site as the DNA of interest (creating sticky ends)
-a gene/sequence of interest is then ligated into the plasmid (vector) using DNA ligase


replication of recombinant plasmids in bacteria
name of process
selection of transformed bacteria

-bacterial cells can be manipulated to take up recombinant plasmids via transformation
-each cell will only contain one recombinant plasmid
-these egineered plasmids typically confer antibiotic resistance therefore there can be single out by subjecting the culture to this antibiotic


DNA libraries

The entire genome from an organism or a tisue can be fragmented then cloned into a vector and transformed. This creates a large poplulation of bacteria which harbors all of your fragmented DNA.
genomic libraries and cDNA libraries (derived from RNA then reverse trancribed)


Use of cloned genes, what they can be used for

when a gene is cloned, adequate amounts of DNA are made to do sequencing
these fragments can also be used as probes in southern and northen blots
can also be used to make proteins using specific expression vectors


Sanger Method of DNa sequencing
what it uses

-uses dideoxy-NTP chain terminator which lack 2' and 3' hydroxyls
-these are added along with dNTPs and during longation have a predetermined chance of stoping an elongation reaction


Next Generation Sequencing

-bases of a small fragment of DNA are sequentially identified from signals emitted as DNA fragments are resynthesized
-this is done across millions of reactions, on a solid platforms in a massively parallel fashion allowing rapid sequencing