CLINING PERMITS PRODUCTION OF MULTIPLE COPIES OF A SPECIFIC GE OR DNA SEGMENT
CLONING PROCESS
POLYMERASE CHAIN REACTION(PCR) COPIES SPECIFIC DNA SEQUENCE
REACTIONS THAT CAN AMPLIFY TARGET DNA SEQUENCES PRESENT IN VERY SMALL QUANITIES
PCR REQUIRES
TWO OLIGONUCLEOTIDE PRIMERS
ONE COMPLEMENTARY TO THE 3' END OF ONE STRAND THE DNA TO BE AMPLIFIED
ONE COMPLEMENTARY TO THE 3' END OF THE OTHER STRAND
PCR
PRIMERS ANNEEAL TO DENATURE DNA/ COMP STRANDS ARE SYNTHESIZED BY HEAT STABLE DNA POLYMERASE
3 STEPS PCR
DENATURATION
ANNEALING
EXTENSION
GENE CLONING
methods for preparing well defined/gene size pieces of dna in multiple identical copies
BACTERIAL RESTRICTION ENZYMES
CUT NA MOLECURES AT A LIMITED NUMBER OF SPECIFIC DNA SEQUENCES CALLED RESTRICTION SITES
USUALLY MAKE MANY CUTES IN DNA MOLECULE-YIELDING A SET OF RESTRICTION FRAGMENTS
MOST USEFULL RESTRICTION ENZYME CUT
STAGGERED WAY- PRODUCING FRAGMENTS WITH STICKY ENDS THAT CAN BOND WITH COMPLEMENTARY STICKY ENDS OF OTHER FRAGMENTS
DNA LIGASE
ENZYME THAT SEALS THE BONDS BETWEEN RESTRICTION FRAGMENTS
PROCESS
ENZIMES CUT AT RESTRICTION SITE
DNA FRAGMNT FROM ANOTHER ORGANISM IS ADED BASE PAIRING ON STICKY ENDS PRODUCE VARIOUS COMBINATION
DNA LIGASE SEALS THE STRANDS
GEL ELECTROPHORESIS
SEPERATES DNA RESTRICTION FRAGMENTS OF DIFFERENT LENGHTS
LONGER(SLOWER FRAGMENTS) TOP
SHORTER(FASTER FRAGMENTS) BOTTOM
RESTRICTION FRAGMENT ANALYSIS
USEFUL FOR COMPARING TWO DIFFERENT DNA MOLECULES SUCH AS 2 ALLELES FOR A GENE
LIBRARIES
PCR CAN CLONE SHORTSEGMENTS OF DNA BUT NOT ENTIRE GENOMES
GENOMIC LIBRARY CONTAIS AT LEAST ONE COPY OF ALL SEQUENCES IN THE GENOME
LIBRARIES CAN BE MADE BY CUTTING GENOMIC DNA WITH RESTRICTION ENZYMES AD LIGATING FRAG INTO VECTOR
LIBRARIES-FLOW CYTOMETRY
INVOLVES MARKING THEN SORTING CHROMOSOMES
SUBGENOMIC FRACTIONS SUCH AS SINGLE CHROMOSOMES CAN BE SORTED USING THIS TECHNIQUE
LIBRARY-CDNA
CONTAINS COMPLEMENTARY DNA COPIES MADE FROM MRNAS
REPRESENTS GENES THAT ARE TRASNCRIPTIONALLY ACTIVE AT THE TIME THE CELLS WERE COLLECTED FROM MRNA ISOLATION
CDNA LIBRARY PREPARED BY:
ISOLATING MRNA FROM CELLS
SYNTHESIZING COMP. DNA USING
REVERSE TRANSCRIPTASE
CLONING CDNA MOLECULES INTO VECTOR
LIBRARIES- CLONES CAN BE RECOVERED
2 MAIN TECHNIQS
SCREENING
MAPPING
SCREENING LIBR.
PROBES COMP. TO PART OF A GENE ARE USED TO SCREEN A LIBR. TO RECOVER CLONES OF A SPECIFIC GENE
SCREENING LIBR PROCESS
CLONES GROWN ON AGAR PLATES TO PRODUCE COLONIES
COLONIES SCREENED BY TRASFERRING BACTERIAL COLOLINES FROM PLATE TO FILTER AND HYBRIDIZIN FILTER WITH A NUCLEIC ACID PROBE TO THE DNA SEQUENCE OF INTEREST
COLONY CORRESPONDING TO THE ONE ON THE PROBE IDENTIFIED ON THE FILTER IS IDENTIFIED AND RECOVERED
LIBRARY MAPPING
A RESTRICTION MAP ESTABLISHES THE NUMBER AND ORDER OF RESTRCITION SITES AND THE DISTANCE BETWEEN RESTRICTION SITES ON A CLONED DNA SEGMENT
SEQUENCING DNA
MOST COMMON METHOD OF DNA SEQUENCING IS DIDEOXY CHAIN TERMINATIO SEQUENCING DEVELOPED BY SANGER
SEQUENCING DNA PROCESS
DNA SEQUENCES IS DENATURE INTO SINGLE STRANDS
SINGLE STRANDS ARE MIXED WITH PRIMERS THAT ATTACH TO THE 3' END OF DNA
SAMPLES GO INTO 4 TUBES W/ DATP DGTP DCTP OR DTTP
TUBES LABELED DDEOXYNUCLEOTIDE (HAVE 3 OH)
DNA SYNTHESIS TAKES PLACE AND PRIMES ARE EXTENDED
HUMAN GENE THERAPY
ALTERATION OF AN AFFICTED INDIVIDUALS GENES
"NORMAL" NON DISEASE GEN IS INTRODUCED INTO THE AFFECTED INDIVIDUALS GENOMES USING A VECTOR
HOLDS GREAT POTENTIAL FOR TREATING DISORDERS TRACEABLE TO A SINGLE DEFECTIVE GENE
DNA FINGERPRINT
SPECIFIC PATER OF BANDS OF RFLP MARKERS ON A GEL
ANIMAL HUSBANDRY/PHARM ANIMALS
TRANSGENIC ANIMALS CONTAIN GENES FROM OTHER ORGANISMS
TI PLASMID
IS THE MOST COMMONLY USED VECTOR FOR INTRUDUCING NEW GENES INTO PLAN CELLS