genetics lect 2 2011 colour 2 slides per page

8/13/2019 Genetics Lect 2 2011 Colour 2 Slides Per Page

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Lecture2

LECTURE 2

Genetics & DNA technology II

BIOSCI101Essential

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

hybridisationiology

4. Amplification of DNA by PCR and gel

electrophoresis

5. Applications of PCR technology

Lecture2

1. Construction of a genomic library

Instead of focusing on a single gene the cloning

procedure starts with a mixture of fragments from theBIOSCI101Essential

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 ofiology

genes.

In addition to plasmids, bacterial artificial

chromosomes (BACs) are also commonly used as

cloning vectors for the construction of libraries.


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Lecture2

Genomic libraries (continued)

Foreign genome

Cut with restriction enzymes into either

small largeor Bacterial artificialBIOSCI101Essential

fragments fragments

Recombinantplasmids

(b) BAC clone

chromosome (BAC)

Largeinsertwithmanygenes

iology

Plasmidclone

(a) Plasmid libr ary (c) Storin g genome libraries

Lecture2

Genomic libraries (continued)

Libraries usually contain a selection of large fragments

BIOSCI101Essential

Plasmid libraries

Relatively small inserts (< 10 kb)

Therefore need a lot of clones

Stored as DNA or cells

iology

Bigger inserts (100300kb)

Need less clones

Stored as DNA or as cells in a multiwell plate


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Lecture2

Construction of a genomic libraries

1. DNA is isolated from an organism and cut into

thousands of pieces with restriction enzymes.BIOSCI101Essential

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.

4. The recombinant plasmid or BAC DNA is used toiology

transform bacterial cells.

5. The collection of the thousands of clones of bacteriaor BACs is a genomic library.

Lecture2

2. Construct ing a cDNA l ibrary

cDNA library - collection of clones containing all of the

gene sequences that are expressed in a particularBIOSCI101Essential

tissue type.

cDNA library is made from mRNA molecules.

Only contains genes that are being expressed at thetime of RNA extraction e.g. tissue specific or conditions ecific.

iology Less complex than genomic library.

Will contain no promoters, untranslated regions orintrons.


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Lecture2

Making complementary DNA (cDNA)

DNA in

nucleusmRNAs incytoplasm

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mRNA

Reversetranscriptase Poly-A tail

DNAstrand

Primer

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55

33

33

A A A A A A

A A AA A A

T T T T T

T T T T T

iology

DNApolymerase

cDNA

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55

33

33

Lecture2

Genomic vs cDNA libraries

If you want to clone a gene but are unsure in what type

of tissue it is expressed - genomic library.BIOSCI101Essential

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 librar is useful for stud in the enesiology

responsible for specific functions e.g. liver or brain.

Changes in patterns of gene expression duringdevelopment - cDNA library.


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Lecture2

3. Screening libraries by hybridisation

DNA hybridisation allows us to sort through the

thousands of clones in a library and find a gene ofBIOSCI101Essential

interest.

We can detect the gene of interest by its ability to

basepair with a complementary sequence of a

another DNA molecule.

iology

fragments with the similar sequence as a gene of

interest.

The probe is tagged with a radioactive or a

fluorescent label.

Lecture2

DNA probe

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iology

After separating the ds target DNA the ss probe will

hydrogen bond to the complementary sequence.


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Lecture2

Nucleic acid probe hybridisation

Radioactivelylabeled prob e

TECHNIQUE 5 3CTCATCACCGGC

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molecules interest

ProbeDNA

Single-strandedDNA fromcell

Film

Multiwell platesholding libraryclones

53

iology

Location ofDNA with thecomplementarysequence

Nylonmembrane

Nylon membrane

Lecture2

4. Polymerase Chain Reaction (PCR) &

gel electrophoresis

PCR is a three step process that produces millions of

copies of a targeted region of DNA.BIOSCI101Essential

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 double-iology

stranded 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)


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Lecture2

Polymerase chain reaction

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iology

Lecture2

Polymerase chain reaction

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iology


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Lecture2

Gel electrophoresis

Separates macromolecules (DNA or proteins) on thebasis of their rate of movement through a gel in an

-BIOSCI101Essential

.

The rate of movement depends on size, electrical

charge, and other physical properties of the

macromolecules.

DNA separation depends mainly on size (length ofiology

ragmen w onger ragmen s m gra ng ess a ong

the gel.

Size markers, DNA of known size are used for

calibration.

Lecture2

Gel electrophoresis (continued)

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iology


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Lecture2

Gel electrophoresis

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iology

A DNA-binding dye fluoresces pink in ultraviolet.

genetics lect 2 2011 colour 2 slides per page

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