????????? Genetic Recombination and Genetic Engineering ????:??? ?????????????

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?????????Genetic Recombination and Genetic
Engineering????????????????????
????
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??????DNA????????????? DNA Recombination and
Gene Transfer Occur Frequently in Nature
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DNA??( DNA recombination )
DNA recombination refers to the process that a
DNA segment moves from one DNA molecule to
another DNA molecule. The following three types
are most commonly observed Homologous
recombination(????)It occurs between two
homologous DNA molecules, also called DNA
crossover. Site-specific recombination(??????) It
occurs at a specific DNA sequence which is
present in both non-homologous DNA molecules that
may have the recombination. Transpositional
recombination(????) A mobile element is inserted
into a target DNA.
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???????????DNA????

• ????(homologous recombination,HR)
  ?????????????,???????????,???DNA??????????????????
  ????????(general recombination)?
• ?????????DNA??,???????????????????

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Homologous recombination occurs between two
homologous DNA molecules. During meiosis, two
homologous pairs of sister chromatids align side
by side. The DNA crossover is very likely to
occur. It could be as often as several times per
meiosis.
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The mechanism of homologous recombination was
first explained by Robin Holliday in 1964.
Holliday???,??????4?????

• ? ???????DNA????
• ? ??DNA????????????DNA??????,??Holliday???
• ? ????????????DNA
• ? Holliday????????,?????????DNA,???

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???
DNA??
????
???
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?????
?????
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????? (????????) ???????????????,???????
?????,?????????DNA?
?????(????????) ????????????,????????????
?????DNA?
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Homologous Recombination Method(Knockout Mouse)
Figure 1. Diagram of gene targeted for
replacement by an engineered construct. The
coding sequence is illustrated by the box with
flanking upstream and downstream DNA sequences
provided. The arrows pointing away from the
targeted gene represent the continuous
chromosomal DNA.
Design and fabricate the DNA construct you want
to insert into the chromosome in place of the
wild-type allele.
Fiugre 2. Diagram of engineered construct that
will be used to replace the wild-type allele. The
upstream and downstream flanking DNA sequences
are identical to those which flank the targeted
locus. The negative marker tk is shown in to the
right of the region of sequence similarity.
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Homologous Recombination Method(Knockout Mouse)
If the targeting vector aligns in a
non-homologous region of the genome, then
recombination is random and the negative
selection marker may become incorporated into the
genome
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?????????????????????

• ??????,???DNA?????????????????????(site specific
  recombination)???,????DNA?????????????????????????
  ?
• ???????????,????????DNA??,?????????DNA???????????
  ??????

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(?)????DNA??? ????????????????????????????????
??????????????????????cDNA????????(long
terminal repeat, LTR)?
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excisionase
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(?)?????????
????H???????????
hix
hix
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hix???????,?????H????Hin???????????(??)?H?????????
P,????hin????,???????H2?rH1????,??(??)?H2?rH1????r
H1?H1????????
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(?)??????????
?????(Ig),?????(L?)?????(H?)??,?????????????,?????
???(???),???????
???????
???????
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CACAGTG (12/23) ACAAAAACC
GTGTCAC TGTTTTTGG
??????
????
??(IgH)???V-D-J?????(IgL)???V-J?????????????V?????
,J???????D?????????????????(recombination signal
sequence, RSS)??????????rag (recombination
activating gene)????,???????RAG1?RAG2?
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??????????????????DNA??

• ?????????????????,??????????????????????????DNA???
  ?????????(transposon, Tn)????????????????????????(
  transposition),??????(transpositional
  recombination)?

A transposon (transposable element) is a DNA
sequence able to insert itself (or a copy of
itself) at a new location in the genome, without
having any sequence relationship with the target
locus.
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(?)????
Insertion sequences are simple transposition
modules
????(insertion sequences, IS)??
inverted repeats (IR)941bp transposase
gene repeated sequences412bp
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(?)???
??????????? ??????? ???????????
???????IS?? ????????????????,???????(???)?????????
Tn?????????????????????????????(arms)???????(compo
site elements)?
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???? ?????(conservative transposition)
?????(duplicative transposition)
Conservative transposition describes the movement
of a transposon that leaves a donor site (usually
generating a double-strand break) and moves to a
new site.
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duplication transposition
insertion sequences
target
transposase
transposase
5
transposase
3
3
transposase
3
5
target
transposase
5
3
3
transposase
3
5
Duplication transposition describes the movement
of a transposon by a mechanism in which first it
is replicated, and then one copy is transferred
to a new site.
polymerase I ligase
transposase
transposase
transposase
transposase
transposase
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? ? ? ??DNA??
DNA Recombination Technique
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??????

• ??????
• DNA??
• ???
• ????
• ????
• ???????????

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????DNA??????
(?) DNA??
???????,????????????????DNA?????????????DNA?????
(replicon),?????????????,???????????????,?????????
????DNA??,?????????DNA (recombinant DNA) ?
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?????? ?????????????????????
????(genetic engineering) ?????????????????,???
?DNA???
?? ? ???????????DNA ? ???????????(???)
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(?)???

• ????????
• DNA????
• ????
• T4 DNA???
• ?????
• ?????
• Taq DNA???

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??DNA?????????
? ? ? ? ?
???????? ??????,??DNA
DNA??? ??DNA????5????3?????????????,?DNA????????DNA???????
DNA???? ????cDNA??????? ??????????? DNA???? ??3??
Klenow?? ??DNA???I???,????DNA???I?5??3????3??5?????,??5??3?????????cDNA?????,??DNA 3??????
???? ??cDNA ??DNA???I????,???DNA????
??????? ???????5???????,?????
????? ?3?????????????
????? ???????
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????????
(restriction endonuclease) ??DNA?????,
??????????????DNA?????????
Bam H?

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?? ?????? (?????????????)
?? ??????????????????,????DNA, ????DNA?
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??
Hin d?
Haemophilus influenzae d? ??????d??????
? ? ? ?
????????????????,??? ???????????????,??? ???????
? ???????????????
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?????????
????(palindrome)
???????????
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Hind?
Bam H?


????
????
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???????????????????,???DNA?,?????????,????????????
??????????(compatible end)?
Bg l?
Bam H?


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DNA???????DNA????
DNA???(DNA ligase)???????3'-OH?5'-??????3',
5'-?????,???DNA?????????????????
????????
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???????????????????
???????(bacterial alkaline
phosphatase,BAP)????(calf intestinal alkaline
phosphatase,CIP)?????DNA(RNA)5'???????,?5'-P??5'-O
H,???????????????
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?????RNA?????cDNA
RNA
RNA
5'
3'
5'
3'
AAAAAA
???? (reverse transcriptase)
Oligo-dT
????
AAAAAA
TTTTTT
cDNA
cDNA
???????cDNA??
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Taq DNA??????PCR

• Taq DNA?????????????????,???PCR?????5'?
  3'??????5'?3'?????,???3'?5'?????,???3'?5'????,??PC
  R???????????,??????????????,?????????????DNA???,??
  ???PCR??????????
• ??,Taq DNA????????????,?????DNA??3'???
  ???????????(A)????PCR????????3'-T??????(T??)??(?T-
  A??)?

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(?)????
source of the target gene It is from genomic
DNA directly, this is prokaryotic gene only
generally. It is from artificial synthesis,
this is simple polypeptide gene generally.
It is from mRNA. It is from genomic
library or cDNA library. Polymerase Chain
Reaction (PCR).
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?????
?????????????DNA??
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cDNA library
transformation
cDNA library
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??????(PCR)
????????????????????????DNA?cDNA?????,??????????
?? ?????????????,???????PCR??,????DNA?cDNA
???????,?????????DNA???????
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ing
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(?)????
?? ???????,???????????????????????DNA???
???? ??DNA ???DNA ??DNA
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????(cloning vector) ???????DNA???????????????????
? ????(expression vector) ???????DNA????????????
??????????????
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???????

• ?????
• ????????????,???????????
• ?????(??DNA???),???????????,???????
• ????,????????DNA?

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?? (plasmid)
?????????????DNA???
?? ?????????????????????, ??????????????
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????DNA??????
???????
??????????
??????????
??DNA?????
??????
???????
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separate target gene
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cut and ligate target gene and vector
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a ?????
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??DNA?????????
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???????
??????? ??????? ??????? ?????????
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1. ?????? (E.coli????????) ?????? ????
?????? ??????? E.coli???????
?????????DNA ???????????? ???????????????
(inclusion body) ???????????
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2. ?????? ?????????????
????????cDNA??????DNA ??????????? ????????? ????
?????????
?? ??????????????
??????? DEAE??????? ??? ????? ????
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(?) ????????????????????
?????????,???????????????,???????????????????,????
??????????????????????????????????????????????????
????
R???? P??? SDSD??TT??????
????????
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1. ?????????????????,???RNA??????????????????????
????

• Lac???(?????)
• Trp???(??????)
• Tac???(????????????)
• PL?PR???(?????????????)
• T7???

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2. SD??????????? mRNA??????????????????
?(ribosome binding site)????SD??(Shine-Dalgarno
sequence)??????????813 bp?,?????????,??????30S???
??16S rRNA 3'???????????SD???????????,????????????
??
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3. ?????????????????? ?????????????,??????
????,?????????????,???????????????????
??????????,??????bp,??????bp?
??????????RNA??????????????,?????RNA???,??RNA????
?????????????????????????,???????????????
?????????????????????????
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4. ??????????? ???????????,??????????
(1)???????? (2)??????? (3)???????
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(?)?????????????????????
????????????????????????????????????????
?????????? ? ?????????????????????????poly A
????? ? ?????????? ? ??????????
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???????????
OriPro????????P???MCS?????TT??????orieuk?
?????????????????????????
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????????????????????????????????????????,?????????
??,?Rous??????????????(long terminal
repeats,LTR)?SV40??????????????????????(CMV)??????
???????????????????????,???????????????
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• ?????????poly A ???????????mRNA??????poly A?
• ?mRNA??poly A???mRNA 3'???AAUAAA?????GU?U????
• ????cDNA???????AAUAAA?????poly A,???????????poly
  A????????,???????poly A ?????
• ?????SV40?poly A?????237 bp,????????????????????po
  ly A ????????????????,????????DNA??,?mRNA????????

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???????????,???????????
?????? ?????? ?????????
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????????????
(?)?????????? ???????????????,???????????
(?)????
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??DNA????
? ? ? ?
????????? ??
????VIII ????
????-?????????? ???????
??????? ???????
????(bFGF, EGF) ?????????
??? ?????
??? ?????
???(? 1b, ?2a, ? 2b, ?) ??????????
????? ??????????
??????? ?????
????? ?????????????????????
????(CHO, ??) ????
????B????????? ????
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(?)????
????(genetic testing)???????????????????,?DNA?????
????????????????????????

• carrier screening, which involves identifying
  unaffected individuals who carry one copy of a
  gene for a disease that requires two copies for
  the disease to be expressed
• preimplantation genetic diagnosis
• prenatal diagnostic testing
• newborn screening-phenylketonuria, congenital
  hypothyroidism
• Genealogical DNA test (for genetic genealogy
  purposes)
• presymptomatic testing for predicting
  adult-onset disorders such as Huntington's
  disease and Alzheimer's disease
• confirmational diagnosis of a symptomatic
  individual
• forensic/identity testing

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????
????????DNA??
?????DNA???
?? . ???????? . ???????????? .
??????,???DNA??? . ?????????,????????????
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(?)????
?? ????(gene therapy)???????????????????,?????????
???,??????????
?? ??????? (somatic cell gene therapy) ???????
(germ line gene therapy)
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???? Gene therapy
The most common form of gene therapy
involves using DNA that encodes a functional,
therapeutic gene in order to replace a mutated
gene. Other forms involve directly correcting a
mutation, or using DNA that encodes a therapeutic
protein drug (rather than a natural human gene)
to provide treatment.
Although early clinical failures led many
to dismiss gene therapy as over-hyped, clinical
successes in 2009-2011 have bolstered new
optimism in the promise of gene therapy. These
include successful treatment of patients with the
retinal disease Leber's Congenital Amaurosis,
X-linked SCID, ADA-SCID, adrenoleukodystrophy,
and Parkinson's disease.
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???? Gene therapy-problems
Short-lived nature of gene therapy Problems with
integrating therapeutic DNA into the genome and
the rapidly dividing nature of many cells prevent
gene therapy from achieving any long-term
benefits. Patients will have to undergo multiple
rounds of gene therapy. Immune response The risk
of stimulating the immune system in a way that
reduces gene therapy effectiveness is always a
possibility. Furthermore, the immune system's
enhanced response to invaders that it has seen
before makes it difficult for gene therapy to be
repeated in patients. Problems with viral vectors
Viruses, the carrier of choice in most gene
therapy studies, present a variety of potential
problems to the patient. Multigene disorders
Multigene or multifactorial disorders such as
heart disease, high blood pressure, Alzheimer's
disease, arthritis, and diabetes, would be
especially difficult to treat effectively using
gene therapy. Chance of inducing a tumor - If the
DNA is integrated in the wrong place in the
genome, for example in a tumor suppressor gene,
it could induce a tumor.
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????? ?????????
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1. ????????
A ????????,???????? B ????????,???????? C
????????,???????? D ????????,???????? E ?????
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2. ??????????????? ?
A ????????? B ????DNA?????I?? C
?????DNA???????? D ?????????? E ???????????????
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3. ???????,???????? ?
A ??????DNA?? B ???2-3kb, ?????kb C
???????????????? D ??????? E ??????????????
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4. ????????DNA??????
A ???????????? B ???????? C ?????????? D
?????? E ???????
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5. DNA????????????

1. ?????????
2. ??????????????????????
3. ????????DNA???DNA,?????
4. ????????????????????
5. ???????????

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6. ????????DNA????????
A ???? B ????? C ????? D DNA???I E DNA???
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7. ???????????????
A ????? B ????? C ?????? D ?????? E ?????
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8. ???DNA?????,????DNA???????
A ?????? B ?????? C cDNA??? D PCR? E
????DNA????
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9. ??????????????????
A DNA??? B RNA??? C DNA??? D RNA??? E
????????
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10. ?????????????????
A E.coli ???? B ?????? C ?????? D ?????? E
?????????
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11. The nucleotide number which restriction
enzyme recognize in DNA nucleotide
sequence is
A 4, 5 or 6 B 5, 6 or 7 C 6, 7 or 8 D 4, 6 or
8 E 4 - 8
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12. The technique used in identification of DNA
is
A northern blotting B southern blotting C
Western blotting D affinity chromatography E
ion exchange chromatography
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13. The way of gene recombination doesnt include
A transformation B transduction C
transposition D change-over?? E integration
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14. The abbreviation of polymerase chain reaction
is
A PRC B PER C PDR D BCR E PCR
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15. ????????,??????????
A ????? B ????? C southern ?? D ?????? E
??????
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16. ?????,????????

A ???? B PCR?? C cDNA?? D ????? E
????????DNA????
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17. ??DNA?????????????????

A ??????? B ??? C ???(??DNA????????) D
????????????????? E ?????
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18. ?????????????????

A ????? B DEAE??????? C ??? D ????? E ????
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19. gene cloning also be called
A DNA recombination B RNA recombination C DNA
cloning D RNA cloning E protein replication
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20. The enzyme tools commonly used in
gene cloning technique are

A restriction enzyme B DNA polymerase I C DNA
ligase D reverse transcriptase E terminal
transferase