Molecular Biology - 11) Molecular Engineering

What are the characteristics of vectors?

- Origins of replication: self-replicating - Restriction sites - Identification markers - resistance to antibiotics Most common vectors originated from E.coli.

What are the two types of gene libraries?

1) Genomic libraries: if the DNA is genomic DNA 2) cDNA libraries: if the DNA is a copy of an mRNA population, that is cDNA. cDNAs represent the transcribed parts of the genome

Describe the screening procedure?

1) Hybridization with a nucleic acid probe (PCR probes) produced by PCR 2) A copy of the position of colonies or plaques on a petri dish is made on the surface of a membrane 3) Incubation in a solution of labelled probe 4) After hybridisation and washing, the location of the bound label is determined 5) Group of colonies/plaques to which the label has bound is diluted and replated in subsequent rounds of screening until an individual clone is obtained. 1/6th of clones in a cDNA expression library will produce their encoded pp. Antibodies to the desired protein can be used to screen the library in a process to similar to plaque hybridization to obtain the particular clone.

Give examples of DNA cloning applications:

1) Identification of genes and derivation of protein sequences 2) Isolation and analysis of gene promoters and other control sequences 3) Investigation of protein/enzyme/RNA function by large-scale production of normal and altered forms 4) Identification of polymorphisms and mutations in humans and other organisms. 5) Biotechnology 6) Engineering animals and plants, and gene therapy 7) Engineering proteins to alter their proteins

Describe the steps for subcloning?

1) Isolation of plasmid DNA containing the cloned sequence of interest 2) Cutting of the plasmid into discrete fragments with restriction endonucleases 3) Separation of the fragments by agarose gel electrophoresis 4) Purification of the desired target fragment 5) Ligation of the fragment into a new plasmid vector, to form a new recombinant molecule 6) Transformation is transfer of the ligated plasmid into an E.coli strain 7) Selection of transformed bacteria 8) Analysis of recombinant plasmids

What are the 5 types of cloning vectors?

1) Plasmid 2) Phage 3) Cosmids 4) Bacterial Artificial Chromosomes (BAC) 5) Yeast Artificial Chromosomes (YAC)

What are the general steps of cloning with any vector?

1) Prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary ends. 2) Ligate the foreign DNA into the vector with the enzyme DNA ligase 3) Introduce the DNA into bacterial cells (or yeast cells for YACs) by transformation. 4) Select cells containing foreign DNA by screening for selectable markers (usually drug resistance). Bacteria which have taken up a plasmid are selected by growth on a plate containing an antibiotic to which the plasmid vector encodes resistance

What are some gene engineering applications?

1) Production of recombinant proteins 2) Genetically modified organisms 3) DNA fingerprinting 4) Diagnostic kits 5) Gene therapy

What are the steps to making a representative genomic library?

1) Purification of genomic DNA 2) Genomic DNA is broken randomly into fragments that are the correct size for cloning into the chosen vector 3) Insertion of sequence of interested into the vector 4) Ligation of recombinant DNA 5) Packaging and propagation to create the library

What are the 7 enzymes used in DNA cloning?

1) Restriction endonucleases 2) Reverse transcriptase 3) DNA polymerase 4) Ligase 5) Terminal transferase 6) RNase A 7) Alkaline phosphatase

What are the steps to making a representative cDNA library?

1) mRNA isolation, purification and fractionation 2) Integrity of an mRNA preparation can be checked 3) Synthesis of cDNA by reverse transcriptase 4) Ligation to vector 5) Packaging and propagation to create the library

What are Gene libraries?

A collection of different DNA sequences from an organism each of which has been cloned into a vector for ease of purification storage and analysis.

What is Reproductive cloning?

A method used to make a clone or an identical copy of an entire multicellular organism

What is recombinant DNA?

A short fragment of a genome containing the gene or other sequence of interest in an autonomously replicating piece of DNA known as a vector.

What is a cloning vector?

A small DNA molecule easily isolated from host cells, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA.

What is terminal transferase?

Adds a number of nucleotides to the 3'-end of linear single or dsDNA or RNA.

What is a plasmid?

An extrachromosomal circular DNA molecule that autonomously replicates inside the bacterial cell. Cloning limit, 100 to 10,000 base pairs, or 0.1-10 kilobases.

What is a cosmid?

An extrachromosomal circular DNA molecule that combines features of plasmids and phage. Cloning limit: 35-50kb

What are BACs?

Bacterial Artificial Chromosomes are based on mini-F plasmids. Cloning limit: 75-300kb

What are Restriction endonucleases?

Bacterial enzymes which cut (hydrolyse) DNA into defined and reproducible fragments. In bacteria, they form part of the restriction-modification defense against foreign DNA. They are the basic tools of gene cloning. They produced cohesive or "sticky" ends.

What is a phage?

Derivatives of bacteriophage lambda. Linear DNA molecules. Cloning limit: 8-20kb

What is RNase A?

Digests RNA but not DNA

What is Molecular (gene) cloning?

Exploit the rapid growth of bacterial cells for producing large amounts of identical DNA fragments, which alone have no capacity to reproduce themselves.

What is Reverse Transcriptase?

Makes cDNA on mRNA template

What is Alkaline phosphatase?

Removes phosphate from 5' ends of double or ss DNA or RNA.

What is the screening procedure for gene libraries?

The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library.

What is subcloning?

The simple transfer of a cloned fragment of DNA from one vector to another. Application: it serves to illustrate many of the routine techniques involved in gene therapy.

What are YACs?

Yeast Artificial Chromosomes are an artificial chromosome that contains telomeres, origin of replication, a yeast centromere, and a selectable marker for identification in yeast cells. Cloning limit: 100-1000kb

What is DNA ligase?

repairs breaks in a dsDNA backbone and can covalently rejoin annealed cohesive ends

What is DNA polymerase?

synthesises new strand DNA, builds phosphodiester bonds