 DNA cloning

Section 1: Gene manipulation (Basic concept & basic techniques) section 2: Cloning vectors (Compare various Cloning vectors) Section 3: Gene libraries & screening (Genomic libraries & cDNA libraries) Section 4: Analysis and uses of a clone (Analysis & Applications)

DNA cloning makes molecular biology possible Molecular biology seeks to explain the relationships between the structure and function of biological molecules (DNA, RNA, protein, other small regulators) and how these relationships contribute to the operation and control of biochemical processes (replication, transcription, translation)

DNA cloning is to place a relatively short fragment of a genome, which might contain the gene or other sequence of interest, in an autonomously replicating piece of DNA, known as a vector, forming RECOMBINANT DNA, which can be replicates independently of the original genome, and normally in other host species. Propagation of the host organism containing the recombinant DNA forms a set of genetically identical organism, or a ClONE. This process is called DNA cloning. What is DNA cloning?

Prepare interested DNA fragment from a genome (insert) Plasmid preparation (vector) Restriction digestion (trimming the DNA ends) Ligation (join the insert and the vector) Transformation & selection (introduce the plasmids into host cells) Assay of the recombinant DNA: (1) is it a right clone? G4 (2) Analyze the biological function of the cloned gene/DNA DNA Cloning: a simplified flow chart I PCR or Genomic DNA

Vector Target DNA Recombinant plasmid/DNA A simple example ofDNA Cloning Clone

Transfer of a fragment of cloned DNA from one vector to another. 1.Enables us to investigate a short region of a large cloned fragment in more detail. 2.To transfer a gene from one plasmid to a vector designed to express it in a particular species. Subcloning

Prepare the interested DNA fragment from cloned DNA (insert) Plasmid preparation (vector) Restriction digestion (trimming the DNA ends) Ligation (join the insert and the vector) Transformation & selection (introduce the plasmids into host cells) Assay of the recombinant DNA DNA subcloning: a flow chart PCR or Plasmid, phage

Hosts Host organism/cell: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning. Prokaryotic host : E. coli (most cases) Eukaryotic host : Yeast (Saccharomyces cerevisiae), mammalian, insect

General features of a Vector 1.autonomously replicating DNA independent of host’s genome, thus contains origin sequence. 2.Easily to be isolated from the host cell 3.Most are circular, some are linear 4.Contains at least one selective marker, which allows host cells containing the vector to be selected among those which do not. 5.Contains a multiple cloning site (MCS) vectors

Types of vectors Cloning vectors Expression vectors Integration vectors

Cloning vectors: allowing the exogenous DNA to be inserted, stored, and manipulated at DNA level. E. coli cloning vector: plasmids, bacteriophages (l and M13), plasmid- bacteriophage l hybrids (cosmids), BAC. Yeast cloning vector: yeast artificial chromosomes (YACs)

Expression vectors: allowing the exogenous DNA to be inserted and expressed. Promoter and terminator for RNA transcription are required. RBS (E. coli), start and stop codons are required for protein synthesis.

Amp r ori pUC18 (3 kb) MCS (Multiple cloning sites, 多科隆位点） Lac promoter lacZ’ …ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATG CA…. T h rA s n S er S e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser… EcoRISacIKpnI SmaI XmaI BamHI XbaI SalI HincII AccI PstISphI Lac Z

1.The ORF of the inserted gene has to be in the same direction as that of the lacZ 2.A fusion protein contains the N-terminal sequence of lacZ and the inserted ORF will be produced

T7 promoter RBS Start codon MCS Transcription terminator Amp r ori T7 expression vector back

MCS Yeast expression vectors:

Inserts DNA cloning: 1.PCR amplification of the interested DNA and restriction 2.Genomic DNA preparation and fragmentation (restriction sites addition) 3.mRNA isolation, cDNA preparation (restriction site) DNA subcloning: 1.PCR amplification of the interested DNA and restriction 2.Plasmid/recombinant DNA preparation and restriction

DNA libraries DNA librariesDNA libraries are sets of DNA clones, each of which has been derived from the insertion of a different fragment into a vector followed by propagation in the host. A clone is a genetically distinct individual or set of identical individuals Genomic librariescDNA libraries

Genomic libraries prepared from random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non- coding DNA cDNA libraries DNA copies (cDNA) synthesized from the mRNA by reverse transcription are inserted into a vector to form a cDNA library. Much more efficient in identifying a gene, but do not contain DNA coding functional RNA or noncoding sequence.

Hybridization to identify the interested DNA or its RNA product 1.Radiolabeled probes which is complementary to a region of the interested gene Probes: An oligonucleotide derived from the sequence of a protein product of the gene A DNA fragment/oligo from a related gene of another species 2.Blotting the DNA or RNA on a membrane 3.Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane Screening libraries (I3)

Identify the protein product of an interested gene, called expression screening 1.Protein activity 2.Western blotting using a specific antibody

 核酸分子杂交（ DNA/DNA or DNA/RNA ） 技术 核酸分子杂交技术，是在 1968 年由华盛顿卡 内基学院（ Cavnegie Institute of Washington ） 的 Roy Britten 及其同事发明的。所依据的原理 是，带有互补的特定核苷酸序列的单链 DNA 或 RNA ，当它们混合在一起时，其相应的同源区 段将会退火形成双链的结构。 核酸分子杂交技术，是在 1968 年由华盛顿卡 内基学院（ Cavnegie Institute of Washington ） 的 Roy Britten 及其同事发明的。所依据的原理 是，带有互补的特定核苷酸序列的单链 DNA 或 RNA ，当它们混合在一起时，其相应的同源区 段将会退火形成双链的结构。

萨瑟恩 DNA 印迹杂交 萨瑟恩 DNA 印迹杂交 根据毛细管作用的原理，使在电泳凝胶 中分离的 DNA 片段转移并结合在适当的滤膜 上，然后通过同标记的单链 DNA 或 RNA 探 针的杂交作用检测这些被转移的 DNA 片段， 这种实验方法叫做 DNA 印迹杂交技术。由于 它是由 E.Southern 于 1975 年首先设计出来的， 故又叫 Southern DNA 印迹转移技术。 根据毛细管作用的原理，使在电泳凝胶 中分离的 DNA 片段转移并结合在适当的滤膜 上，然后通过同标记的单链 DNA 或 RNA 探 针的杂交作用检测这些被转移的 DNA 片段， 这种实验方法叫做 DNA 印迹杂交技术。由于 它是由 E.Southern 于 1975 年首先设计出来的， 故又叫 Southern DNA 印迹转移技术。

（a）（a） （b）（b） （c）（c） （d）（d） （e）（e） 基因组 DNA DNA 限制片段 硝酸纤维素滤膜 同探针同源杂交 的基因 DNA 片段 X 光底片 Southern 凝 胶转移杂交 技术

诺赛恩 RNA 印迹技术（ Northern blotting ） 诺赛恩 RNA 印迹技术（ Northern blotting ） 1979 年， J.C.Alwine 等人发展而来，是将 RNA 分子从电泳凝 胶转移到硝酸纤维素滤膜或其他化学修饰的活性滤纸上，进行 核酸杂交的一种实验方法。由于这种方法与萨瑟恩 DNA 印迹杂 交技术十分类似，所以叫做诺赛恩 RNA 印迹技术（ Northern blotting ）。 1979 年， J.C.Alwine 等人发展而来，是将 RNA 分子从电泳凝 胶转移到硝酸纤维素滤膜或其他化学修饰的活性滤纸上，进行 核酸杂交的一种实验方法。由于这种方法与萨瑟恩 DNA 印迹杂 交技术十分类似，所以叫做诺赛恩 RNA 印迹技术（ Northern blotting ）。 而将蛋白质从电泳凝胶中转移到硝酸纤维素滤膜上，然后同 放射性同位素 125 I 标记的特定蛋白质之抗体进行反应，这种技术 叫做韦斯顿蛋白质杂交技术（ Western blotting ）。 而将蛋白质从电泳凝胶中转移到硝酸纤维素滤膜上，然后同 放射性同位素 125 I 标记的特定蛋白质之抗体进行反应，这种技术 叫做韦斯顿蛋白质杂交技术（ Western blotting ）。

检测重组体克隆的菌落杂交技术 菌落杂交 菌落杂交