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21.1.1

RECOMBINANT DNA = COMBINING FRAGMENTS OF DNA FROM TWO SPECIES RESULTING IN TRANSGENIC ORGANISM OR GENETICALLY MODIFIED ORGANISM

RECOMBINANT DNA TECHNOLOGY = TRANSFER OF DNA FROM ONE SPECIES TO ANOTHER RESULTING IN TRANSLATION WITHIN THE RECIPIENT DUE TO UNIVERSAL NATURE OF THE GENETIC CODE. 1 = ISOLATION OF GENE OF INTEREST, 2 = INSERTION OF DNA INTO VECTOR (CARRIER), 3 = TRANSFORMATION OF DNA INTO SUITABLE HOST CELL, 4 = IDENTIFICATION OF HOST CELLS THAT HAVE TAKE UP THE GENE, 5 = GROWTH OR CLONING OF THE HOST CELLS

REVERSE TRANSCRIPTASE = CONVERTS mRNA TO cDNA, 1 = A CELL THAT READILY PRODUCES THE PROTEIN IS SELECTED AND THE RELEVANT mRNA IS EXTRACTED, 2 = REVERSE TRANSCRIPTASE MAKES cDNA FROM mRNA USING COMPLEMENTARY NUCLEOTIDES, 3 = DNA POLYMERASE BUILDS UP COMPLIMENTARY NUCLEOTIDES ON THE cDNA TEMPLATE TO CREATE A DOUBLE STRAND CONTAINING THE GENE, 4 = INTRONS MUST BE REMOVED OR PROTEIN WOULD BE FAULTY

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21.1.2

RESTRICTION ENDONUCLEASES = ENZYMES THAT CUT DNA AT SPECIFIC BASE SEQUENCES THAT ARE 6 BASES LONG AND PALINDROMIC, PRODUCE BLUNT AND STICKY ENDS

GENE MACHINE = MANUFACTURES GENES, 1 = DESIRED NUCLEOTIDE SEQUENCE INPUT INTO COMPUTER AND CHECKED FOR BIOSAFETY, 2 = COMPUTER DESIGNS SMALL OVERLAPPING SINGLE STRANDED DNA CALLED OGLIONUCLEOTIDES, 3 = OGLIONUCLEOTIDES ARE SYNTHESISED AND ASSEMBLED, 4 = OGLIONUCLEOTIDES JOINED TO MAKE A GENE WITH NO INTRONS, 5 = GENE REPLICATED USING PCR, 6 = GENES CLONED BY INSERTING THEM INTO A BACTERIAL PLASMID AND SEQUENCED TO DISCARD ERRORS, 6 = CORRECT GENES TRANSFERRED TO HOST CELL

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21.2

IN VIVO GENE CLONING = USES STICKY ENDS AS MAKES LIGATION MORE SPECIFIC, TO PREPARE DNA FOR INSERTION PLACE APPROPRIATE PROMOTER IN FRONT OF GENE TO ALLOW RNA POLYMERASE TO BIND, PLACE A TERMINATOR BEHIND THE GENE TO TELL RNA POLYMERASE TO DETACH

USE OF A VECTOR = TO TRANSPORT DNA TO HOST CELL, 1 = CUT VECTOR WITH STICKY END RESTRICTION ENDONUCLEASE, 2 = CUT DESIRED DNA QITH SAME RESTRICTION ENDONUCLEASE AND MIX WITH VECTOR, 3 = SINGLE STRANDED DNA OVERHANGS PAIR WITH EACHOTHER, 4 = DNA LIGASE JOINS DNA BACKBONES

TRANSFORMATION OF PLASMID = DNA IN RECOMBINANT PLASMID INSERTED INTO HOST CELL, MIX BACTERIA WITH CALCIUM IONS TO MAKE IT PERMEABLE, GIVE HEAT SHOCK TO 45 DEGREES TO MAKE PLASMID ENTER CELL, NOT ALL PLASMIDS CONTAIN THE DNA

MARKER GENES = USED TO FIND WHICH CELLS CONTAIN PLASMIDS WITH THE GENE, ANTIBIOTIC RESISTANCE, FLUORESCENCE AND ENZYME MARKER GENES USED

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21.3

IN VITRO GENE CLONING = POLYMERASE CHAIN REACTION IS RAPID AND EFFICIENT PRODUCTION OF LARGE NUMBERS OF IDENTICAL COPIES OF A DNA MOLECULE, REQUIRES THE DESIRED DNA FRAGMENT, PAIR OF PRIMERS, NUCLEOTIDES, HEAT STABLE DNA POLYMERASE AND A THERMOCYCLER TO RAISE TEMP PRECISELY

STEPS OF PCR = 1 = STRANDS OF ORIGINAL DNA SEPARATED  BY HEATING TO 95 DEGREES, 2 = RAPIDLY COOL TO 55 ALLOWING PRIMERS TO ANNEAL, 3 = HEAT TO 72, DNA POLYMERASE MAKES TWO NEW STRANDS BY ADDING NUCLEOTIDES TO PRIMER

ADVANTAGES OF IN VIVO = NO RISK OF CONTAMINATION, VERY ACCURATE, PRODUCES LARGE QUANTITIES OF GENE PRODUCTS

ADVANTAGES OF IN VITRO = VERY FAST, DOES NOT REQUIRE LIVING CELLS

ETHICAL/SOCIAL/FINANCIAL ISSUES = USING MICROORGANISMS CHEAPER THAN USING MAMALS, FEWER ETHICAL ISSUES WITH USING MICROORGANISMS

LINK TO GENE THERAPY = SOMATIC CELL THERAPY TARGETS TISSUE GENE NOT INSERTED INTO GAMETE SO FUTURE GENERATIONS CAN STILL INHERIT, GERM CELL THERAPY OCCURS IN FERTILISED EGGS SO ALL BODY CELLS AFFECTED

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21.4

DNA PROBES = SHORT SINGLE STRANDED PIECES OF DNA COMPLEMENTARY TO THE GENE BEING SCREENED FOR USUALLY RADIOACTIVELY LABELLED, 1 = PROBE MADE USING PCR, 2 = DNA TO BE TESTED SEPARATED INTO TWO STRANDS, 3 = PROBE BINDS TO COMPLEMENTARY DNA (HYBRIDISATION), 4 = GENE OF INTEREST LOCATED USING LABEL ON PROBE

GENETIC SCREENING = IDENTIFIES MUTATIONS IN TUMOUR SUPPRESSOR GENES, IDENTIFY HETEROZYGOUS INDIVIDUALS AT GREATER RISK OF A CERTAIN CANCER, ALLOWS PEOPLE TO MAKE DECISIONS ABOUT FUTURE TREATMENT

PERSONALISED MEDICINE = DEVELOPED BASED ON INDIVIDUALS GENOTYPE THAT MAY MEAN A CERTAIN DRUG IS MORE OR LESS EFFECTIVE , CAN TAILOR DRUG DOSAGE AND SELECT SPECIFIC TYPES

GENETIC COUNSELLING = PATIENTS EXPLAINED RISKS OF INHERITED DISORDERS AND ADVISED ON CONSEQUENCES, GIVEN OPTIONS FOR FAMILY PLANNING/IVF

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21.5

GENETIC FINGERPRINTING = ANALYSING CLONED DNA FRAGMENTS TO DETERMINE GENETIC RELATIONSHIPS AND VARIABILITY WITHIN A POPULATION

VARIABLE NUMBER TANDEM REPEATS = CONTAINED IN GENOME, PROBABILITY OF TWO PEOPLE HAVING THE SAME VNTR IS VERY LOW, MORE CLOSELY RELATED THE MORE SIMILAR

GEL ELECTROPHORESIS = EXTRACTION OF DNA, DIGESTION USING RESTRICTION ENDONUCLEASES, SEPARATION OF FRAGMENTS, TRANSFER FRAGMENTS TO NYLON MEMBRANE, HYBRIDISATION OF DNA PROBES AND FRAGMENTS, DEVELOPMENT OF XRAY FILM, RESULTS COMPARED TO KNOWN LENGTHS OF DNA

USES = PATERNITY TESTS, MEASURING VARIATION IN A POPULATION, TESTING FOR GENETIC DISEASES, FORENSIC SCIENCE, CHECKING PEDIGREES

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