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LECTURE 2 Genetics & DNA technology II

Key Concepts 1. The construction of a genomic library 2. The difference between a cDNA library and a genomic library 3. The procedures involved in screening libraries by hybridisation 4. Amplification of DNA by PCR and gel electrophoresis 5. Applications of PCR technology
Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential B Biology

1. Construction of a genomic library


Instead of focusing on a single gene the cloning procedure starts with a mixture of fragments from the entire genome - shortgun approach. Genomic library - collection of clones that together contain an entire genome of an organism. The library can be saved and used as a source of genes. In addition to plasmids, bacterial artificial chromosomes (BACs) are also commonly used as cloning vectors for the construction of libraries.

Genomic libraries (continued)


Foreign genome Cut with restriction enzymes into either small large or acte a a artificial t ca Bacterial fragments fragments chromosome (BAC) Large insert with many genes Recombinant plasmids (b) BAC clone

Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential B Biology

Plasmid clone

(a) Plasmid library

(c) Storing genome libraries

Genomic libraries (continued)


Libraries usually contain a selection of large fragments Plasmid libraries Relatively small inserts (< 10 kb) Therefore need a lot of clones Stored as DNA or cells BAC libraries Bigger inserts (100300kb) Need less clones Stored as DNA or as cells in a multiwell plate

Construction of a genomic libraries


Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential B Biology

1. DNA is isolated from an organism and cut into thousands of pieces with restriction enzymes. 2. Plasmids or BACs are cut with the same restriction enzymes, and mixed with the genomic DNA fragments 3. DNA ligase is added and covalently links the two pieces of DNA together. plasmid or BAC DNA is used to 4. The recombinant p transform bacterial cells. 5. The collection of the thousands of clones of bacteria or BACs is a genomic library.

2. Constructing a cDNA library


cDNA library - collection of clones containing all of the gene sequences g q that are expressed p in a p particular tissue type. cDNA library is made from mRNA molecules. Only contains genes that are being expressed at the time of RNA extraction e.g. tissue specific or condition specific. p Less complex than genomic library. Will contain no promoters, untranslated regions or introns.

Making complementary DNA (cDNA)


DNA in nucleus mRNAs in cytoplasm

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mRNA

Reverse transcriptase 3

Poly-A tail

A A A A A A3 T T T T T 5

DNA Primer strand 5 3


A A A A A A 3 T T T T T 5

5 3 DNA polymerase

5 3

3 5 cDNA

Genomic vs cDNA libraries


If you want to clone a gene but are unsure in what type of tissue it is expressed - genomic library. If you are interested in the regulation of the gene (promoters) - genomic library. If you are interested in the coding sequences of a gene cDNA library. A cDNA library y is useful for studying y g the g genes responsible for specific functions e.g. liver or brain. Changes in patterns of gene expression during development - cDNA library.

3. Screening libraries by hybridisation


Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential Biology

DNA hybridisation allows us to sort through the thousands of clones in a library and find a gene of interest. We can detect the gene of interest by its ability to basepair with a complementary sequence of a another DNA molecule. DNA probe single stranded labelled DNA fragments with the similar sequence as a gene of interest. The probe is tagged with a radioactive or a fluorescent label.

DNA probe

After separating the ds target DNA the ss probe will hydrogen bond to the complementary sequence.

Nucleic acid probe hybridisation


Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential B Biology
TECHNIQUE Radioactively labeled probe molecules l l 5 3 CTCATCACCGGC GAGTAGTGGCCG 5 3 Probe DNA Film Singlestranded DNA from cell

Gene of interest

Multiwell plates holding library clones Nylon membrane

Location of DNA with the complementary sequence

Nylon membrane

4. Polymerase Chain Reaction (PCR) & gel electrophoresis


PCR is a three step process that produces millions of copies of a targeted region of DNA. PCR is based on a heat stable DNA polymerase. The polymerase generates the second strand of DNA from a single-stranded template. DNA polymerases can only extend existing doublestranded regions and therefore require a primer. Reagents in a PCR reaction: 1. heat stable DNA polymerase 2. deoxyribonucleotides 3. two primers (one for each strand)

Polymerase chain reaction

Lecture 2 BIOSCI 101 Essential Biology

Polymerase chain reaction

Lecture 2 BIOSCI 101 Essential Biology

Gel electrophoresis
Separates macromolecules (DNA or proteins) on the basis of their rate of movement through a gel in an electrical field - molecular sieve sieve. The rate of movement depends on size, electrical charge, and other physical properties of the macromolecules. DNA separation depends mainly on size (length of f fragment) t) with ith longer l fragments f t migrating i ti less l along l the gel. Size markers, DNA of known size are used for calibration.
Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential Biology

Gel electrophoresis (continued)

Gel electrophoresis
Lecture 2 BIOSCI 101 Essential Biology

A DNA-binding dye fluoresces pink in ultraviolet.