Lecture 21

• restriction endonucleases

  hydrolytic enzymes: make cuts at distinct sites in DNA

  contain a twofold axis of symmetry

  sequences of two DNA chains are inverted and copied

  cuts are identical for each chain and can be staggered or even
• EcoRI restriction endonuclease

  cleaves between G and A (staggered cut w/ sticky ends of the four bases AATT)

  ends will recognize each other by base pairing two different DNA samples (prepared with EcoRI), stick together at the ends and can be covalently joined w/ ligase enzyme
• southern blotting mechanism

  DNA sample cleaved with a restriction endonuclease

  electrophoresis

  separated DNA transferred to nitrocellulose paper

  sample is hybridized to a radioactive DNA probe

  probe binds to specific sequence of DNA

  wash and place in contact with Xray film

  DNA sample band that is hybridized to the radioactive probe can be identified
• Restriction fragment length polymorphism

  Southern blotting: detect genetic diseases that arise from mutations within genes

  effect the size of restriction fragments and alter the electrophoretic migration of DNA fragment

  detected mutation may cause disease directly or be closely linked to a disease causing mutation
• Dideoxy DNA sequencing scheme

  deoxy-ATP represents about 98-99% of the total “A” trinucleotide, and dideoxy-ATP about 1-2%

  most of the time an “A” would be inserted in the sequence and replication would continue pas "A"

  about 1-2% of the new DNA chains would be terminated at each point where an “A” occurs, because replication cannot continue past the point where a dideoxy-A is inserted
• DNA Synthesis with Dideoxy-TTP Step 4

  dATP diffuse to occupy open site

  A of dATP will H bond with T residue

  3’ H atom of primer cannot carry out a NU attack on P atom of the α-P of the dATP(process of elongation terminates)

  Most of the DNA will continue to be elongated until a dideoxy residue is incorporated
• Elution Pattern from Fluorescent Sequencing Experiment.

  Each base has a different color code corresponding to a different fluorescent dideoxy-NTP derivative

  360 base sequence is displayed

  made the human genome project possible
• Solid Phase Synthesis of DNA Oligonucleotides mechanism

  1st nt is connected to a resin bead for ease of purification

  nt has a free 5’ OH group that reacts with activated deoxynt (deoxyribonucleoside 3’- phosphoramidite)

  P atom is oxidized

  protecting group is removed from new 5’ OH group

  whole cycle can be repeated
• why taq polymerase in PCR

  This is the special component of PCR that allows the system to undergo repeated cycles of heating and cooling. It is a heat stable DNA polymerase, isolated from thermophilic bacteria, that can withstand temperatures up to 95C.
• PCR protocol

  A duplex DNA sample heated to 950C for 15 sec to separate strands

  cooled to 540C, and short DNA primers are hybridized to each end of the gene

  raised to 720C, and Taq DNA polymerase copies each strand

  temp raised again to 95C for strand separation

  repeat 20-30 times
• cohesive end method of DNA ligation

  join diverse DNA strands

  Two DNA strands produced w/ the same endonuclease

  can be joined together by annealing DNA fragments w/ DNA ligase

  sticky ends adhere with base pairing
• orignal plasmid for recombinant technology

  carries a gene for antibiotic resistance

  When plasmid is present in the host organism, an enzyme is produced that inactivates the antibiotic, the host becomes insensitive to that antibiotic
• why screen a genomic library

  to isolate a specific gene from entire genome of an organism

  In order to study a specific gene, it is necessary to insert that gene into a plasmid, free of all the other genetic material in the cell
• first step of studying a human gene

  determine which chromosome pairs carried that gene

  order a genomic library made from that chromosome only (set of plasmids with DNA inserts that spanned the entire chromosome)

  library could have a mixed set of host bacteria w/ plasmids and inserts that encompassed the whole chromosome
• cloning of human insulin gene

  cDNA cloned into plasmid w/ active promotor upstream from cDNA

  plasmid transformed into E.coli, bacteria are grown and plated to give hundreds of single colonies

  colonies synthesize desired protein

  identified by western blotting