This document discusses the techniques of restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR). RFLP involves using restriction enzymes to cut DNA into fragments, which are then analyzed via gel electrophoresis and probing. PCR, developed in 1985, allows for exponential amplification of specific DNA regions using oligonucleotide primers, DNA polymerase, and thermal cycling. The PCR process involves repeated cycles of template DNA denaturation, primer annealing, and fragment extension, resulting in millions of copies of the target DNA. Both RFLP and PCR are useful for analyzing DNA samples.

4. PCRPCRPCRPCR
19851985--1989198919891989
19851985

5. 55XX
RFLPRFLP::
PCRPCR::
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33
RFLPRFLP((Restriction Fragment Length PolymorphismRestriction Fragment Length Polymorphism((
22--Restriction of DNARestriction of DNA..
44--Screening by probeScreening by probe..
RFLPRFLP::

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22--Restriction of DNARestriction of DNA::
DNADNA
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8. Eco RI, RIIEco RI, RII
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5 ….G / AATTC….35 ….G / AATTC….355 ..... .....GAATTC.…3GAATTC.…3
101099
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bpbp
Base pair (bpBase pair (bp((A=T, G C≡A=T, G C≡(.(.
Kilo base pair = 1000 bpKilo base pair = 1000 bp Mega base pair = 1000,000 bpMega base pair = 1000,000 bp
101099
==10001000MbpMbp
10101111
==100000100000MbpMbp
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9. 33
electrophoresis analysiselectrophoresis analysis
44--Screening by probeScreening by probe::
ProbeProbeDNADNARNARNAsequencesequenceDNADNARNARNAprobeprobeDNADNAPlatePlate..
Screening by probeScreening by probe..

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RFLPRFLP::
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33

11. PCRPCR::
PCRPCRDr. Kary Mullis, 1985Dr. Kary Mullis, 1985
19931993
PCRPCR
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Polymerase Chain Reaction (PCRPolymerase Chain Reaction (PCR((PolymerasePolymerase..
DNADNA

13. PCRPCR::
55MgClMgCl22
11--Template DNATemplate DNA..
22--Two oligonucleotide primersTwo oligonucleotide primers..
44--Taq polymeraseTaq polymerase..
33dNTPsdNTPs..

14. 11--Template DNATemplate DNA::
DNADNATemplate DNATemplate DNA."."
22--Two oligonucleotide primersTwo oligonucleotide primers::
PrimerPrimer::DNADNARNARNADNADNADNADNAOHOH--33DNADNA..

15. 33dNTPsdNTPs::
dNTPsdNTPs
44--Taq polymeraseTaq polymerase::
DNA pol. IDNA pol. IE. coliE. coliPCRPCR7272
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Taq polTaq pol..Thermus aquaticusThermus aquaticus

16. PCRPCR::
11--Denaturation (92 – 95) 94°C 1 minDenaturation (92 – 95) 94°C 1 min..
22--Annealing 37°C 30 secAnnealing 37°C 30 sec..
33--Extention 68 - 72°C 1 minExtention 68 - 72°C 1 min..
44--Cycling 30 – 35 cycCycling 30 – 35 cyc....

17. 11--Denaturation (92 – 95) 94°C 1 minDenaturation (92 – 95) 94°C 1 min::
DNADNADNADNA9494
22--Annealing 37°C 30 secAnnealing 37°C 30 sec::
primerprimerDNADNA TemplateTemplate
373730303030––6060G/CG/C ,, A/TA/T."."

18. 33--Extention 68 - 72°C 1 minExtention 68 - 72°C 1 min::
Taq polTaq pol..primerprimerprimerprimerTemplateTemplate7272
44--Cycling 30 – 35 cycCycling 30 – 35 cyc..::
3030––353522nn
DNADNAnn.
electrophoresis analysiselectrophoresis analysis
2244101010261026copies of the original DNAcopies of the original DNA
338820201,048,5761,048,576

20. PCRPCRRFLPRFLP:(:(
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22PCRPCR ((PCR techniquesPCR techniques
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44PCR techniquesPCR techniquesDNADNA..