基因工程chapter1112-2013

1、Part Two and ThreeThe Applications of Gene Cloning and DNA Analysis in Research and BiotechnologyChapter 10.Studying gene location and structure Chapter 11.Studying gene expression and function Chapter 12.Studying genomics and post genomics Chapter 13.Production of protein from cloned genes Chapter

2、14 . Gene cloning and DNA analysis in medicinesChapter 15 . Gene cloning and DNA analysis in agricultureChapter 16 . Gene cloning and DNA analysis in forensicscienceStudying gene expression And function Studying the RNA transcript of a gene Studying the regulation of gene expression Identifying and

3、studying the translation product of a cloned geneThree type oftranscription and translationFigure 11.1Figure 11.1exons5 -UTR3 -UTRFigure 11.1(a) Genes are expressed by transcription and translationTranscriptionlTranslation ofGeneDNA moleculeRNA徘 rotein一一一+ r RNA, tRNA mRNA(b) Some genes contain intr

4、onslntrons1 Transcription才rocessmgDNA moleculePrimary RNA transcript- still contains intransn0tasMature RNA no intronsnletor卜侬(c) RNA transcripts include regions either side of the geneGeneDNAStart signal for transcriptionEnd signal11.1 Studying the RNA transcript of a geneDetecting the presence of

5、transcripts of a gene and determining its nucleotide sequence1. Northern hybridizationIf RNA from different tissues is run in different lanes of the gel, then the possibility that the gene is differentially expressed can be examined.4812 16 20 24 28 32 36 40 44 48h DarknessRSLNorthern hybridization1

6、1.1 Studying the transcript of a cloned gene11.2 Studying the regulation of gene expression11.3 Identifying and studying the translation product of a cloned gene4812 16 20 24 28 32 36 40 44 48h DarknessRSL11.2 Studying the regulation of gene expressionFigure 11.8Possible positions for control sequen

7、ces in the region upstream of a gene.The gene is switched on or off by the attachment of regulatory proteins to the control sequences Positive role or negative roleFirstly, how to obtain the upstream of a gene?11.2.1 The methods of cloning the promoter of a geneUsing PCRAdapter PCR (接头PCR法) Reverse

8、PCR(反向PCR法) Tail PCR(热不对称交错PCR)target DNA (Geno,mie, DNA, cDNA)已UL1) Restr1i ctionEnzyme Digestion 接头PCR法53一5l52) Ligatton1515S2S1知区已知区域C2C13 155玉 一 51C1C23) 1st PCR (Primer CtPrimer S1。l4) 2nd PCR (Primer C2. Primer S2),4昙 反向PCR法Known sequence analysisGenomic DNA digestionLigationPCRGenomic DNAClon

9、ed cDNA sequencePuPdREDownstream sequenceUpstream sequenceREREPuPdPuPdRERE Tail-PCR法 (Termal Asymmetric Interlaced PCR)又叫热不对称交错PCRGenomic walking芢薰i ,-一”-，冒一一 ，一句似检测到或 否 ）利用弓 呻邸 3和心 进行第诏次 比 耟反应_l-_. - (I的屯屯反应，- -，- 一. .l一J 一 一.-.,. 覃欢竺 笾=竺匈团11.2.1 The methods of cloning the promoter of a gene Using

10、PCR Adapter PCR (接头PCR法) Reverse PCR(反向PCR法) Tail PCR(热不对称交错PCR) Promoter probe expression vector半乳糖苷酶，新霉素磷酸转移酶，氯霉素乙酰转移酶，二氢 叶酸还原酶，潮霉素磷酸转移酶，荧光素酶，葡糖醛酸酶11.2.2To determine the possible positions for control sequences in the upstream of a gene1. Identifying protein-binding sites on a DNA molecule(a) Gel

11、retardation of DNA-protein complex (凝胶阻滞检测）(b) Footprinting with DNase I(c) Modification interference assays2. Identifying control sequences by deletion analysis(a) Reporter genes(b) Carrying out a deletion analysis(a)Gel retardation of DNA-protein complex (凝胶阻滞检测）A bound protein decreases themobili

12、ty of a DNA fragment during gel electrophoresis.Fig 11.10Carrying out a gel retardation experiment Extraction of gDNA21乙：343141亡一55 盲一圈Agarose gel22 亡一星Lane 1 : DNA fragment Lane 2 : DNA fragment +bound protei 一 卢气画一-匾 Identifying protein-binding sites on a DNA molecule(a) Gel retardation of

13、 DNA-protein complex (凝胶阻滞检测）(b) Footprinting with DNase I(c) Modification interference assays Identifying control sequences by deletion analysis(a) Reporter genes(b) Carrying out a deletion analysis(b) Footprinting (足迹法）Fig. 11.11A bound protein protects a region of a DNA from degradation b

14、y a nuclease such as DNase IFootprinting(足迹法） Identifying protein-binding sites on a DNA molecule(a) Gel retardation of DNA-protein complex (凝胶阻滞检测）(b) Footprinting with DNase I(c) Modification interference assays Identifying control sequences by deletion analysis(a) Reporter genes(

15、b) Carrying out a deletion analysisEnha.n oerSiIene erDe!,ete th,e enhance rPmmoterGene-.ExpressionsiIeno 可.-.Lsss express ion卡M o r e exp ress iloni .,.The principle behind del etion anal ysis.Reporter genesFig 11.16半乳糖苷酶，新霉素磷酸转移酶，氯霉素乙酰转移酶，二氢 叶酸还原酶，潮霉素磷酸转移酶，荧光素酶，葡糖醛酸酶Deletion analysis by restrictio

16、n enzymeFig 11.17Deletion analysis.A reporter gene has been attached to the upstream region of a seed- specific gene from a plant.Deletion analysis by PCRGhNP3GUS-232 -1GhNP2GUS-617 -1GhNP1GUS-1078 -1GhNP1GhNP211.1 Studying the transcript of a cloned gene11.2 Studying the regulation of gene expressi

17、on11.3 Identifying and studying the translation product of a cloned gene11.3.1 HRT (Hybrid-release translation) and HART (hybrid-arrest translation) can identify the translation product of a cloned gene11.3.2 Analysis of protein functionCell-freeAAtranslation systemsare cell extracts, usually prepar

18、ed from germinating wheat seeds or from rabbit reticulocyte cells and containing ribosomes, tRNAs, and all the other molecules needed for protein synthesis.Fig 11.18 Hybrid- release translationFig 11.19 Hybrid- arrest translationFig 11.20 We can also use the pGEM3Z vector to synthesize RNA in vitroU

19、se these proteins, we can do some experiments, such as pull down, co-immunoprecipitation, etc, to uncover the mechanism of a gene product.11.1 Studying the transcript of a cloned gene11.2 Studying the regulation of gene expression11.3 Identifying and studying the translation product of a cloned gene

20、11.3.1 HRT (Hybrid-release translation) andHART (hybrid-arrest translation) can identify the translation product of a cloned gene11.3.2 Analysis of protein functionUsing mutation in the coding region to determine protein functionFig 11.21Differenttypes of mutagenesis techniquesFig 11.22Fig 11.24Olig

21、onucleotide-directed point mutagenesisFig 11.23important amino acidPhag,e 炟 飞ring们它 mu讫恁 d ge门仑Phaga carrying normal gen它agaPlaqu,e.s con扫inin g订e mu讫11ed ge.n,ePl,aquesPlaque hybridiz ationPmbB with |ab0|ed oliganu1cleotid它Creating a point mutation in a cloned geneFig 11.25One method of using PCR to create a directed mutati