goal of this chapter 20: this chapter is introducing many genetic technologies that you will need to...

Goal of this Chapter 20:

•This chapter is introducing many genetic technologies that you will need to understand.

-Using Vectors -PCR

-Using cDNA

-Transformation/Transduction

-Electrophoresis

Restriction Enzymes

-First discovered in 1960’s…these enzymes naturally occur in bacteria where they protect

against intruding DNA

-protection is restriction…meaning the foreign DNA is cut up in small segments

-most of them recognize only short, SPECIFIC sequences called recognition sequences

-Bacteria prevent their own DNA from getting cut by methylating their own

DNA VectorsYou need some kind of vector (carrier) to move DNA from a test tube into a cell.

-The two most common? PLASMIDS and VIRUSES (especially bacteriophages)

-Thus think about transformation and transduction

Transformation: http://www.learner.org/channel/courses/biology/archive/animations/hires/a_infect3_h.html

Transduction:

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_2.html

http://www.learner.org/channel/courses/biology/archive/animations/hires/a_infect3_h.html

http://www.learner.org/channel/courses/biology/archive/animations/hires/a_infect3_h.html



We need a host!Bacteria (remember: prokaryotes) are often used as hosts

because:

1) Their DNA can be isolated from and reintroduced into bacterial cells

2) They grow quickly and rapidly

Disadvantage:

3) Bacterial cells may not be able to use a eukaryote’s gene since they often use different enzymes

Eukaryotes can be used as hosts, and yeast does quite well. It is very difficult to get plant/animal cells to take up foreign DNA

• One basic cloning technique begins with the insertion of a foreign gene into a bacterial plasmid.

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Fig. 20.1

• The presence of introns creates problems for expressing these genes in bacteria.– To express eukaryotic genes in bacteria, a fully

processed mRNA acts as the template for the synthesis of a complementary strand using reverse transcriptase.

– This complementary DNA (cDNA), with a promoter, can be attached to a vector for replication, transcription, and translation inside bacteria.



Fig. 20.5

• Complementary DNA is DNA made in vitro using mRNA as a template and the enzyme reverse transcriptase.

• Molecular biologists can avoid incompatibility problems by using eukaryotic cells as host for cloning and expressing eukaryotic genes.

• Yeast cells, single-celled fungi, are as easy to grow as bacteria and have plasmids, rare for eukaryotes.

• Scientists have constructed yeast artificial chromosomes (YACs) - an origin site for replication, a centromere, and two telomeres -with foreign DNA.

• These chromosomes behave normally in mitosis and can carry more DNA than a plasmid. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

• In the “shotgun” cloning approach, a mixture of fragments from the entire genome is included in thousands of different recombinant plasmids.

• A complete set of recombinant plasmid clones, each carrying copies of a particular segment from the initial genome, forms a genomic library.– The library can be saved and used as a source of other

genes or for gene mapping.

4. Cloned genes are stored in DNA libraries


• A more limited kind of gene library can be developed from complementary DNA.– During the process of producing cDNA, all mRNAs

are converted to cDNA strands by reverse transcriptase.

– This cDNA library represents that part of a cell’s genome that was transcribed in the starting cells.

– This is an advantage if a researcher wants to study the genes responsible for specialized functions of a particular kind of cell.

– By making cDNA libraries from cells of the same type at different times in the life of an organism, one can trace changes in the patterns of gene expression.


How cDNA is Made

• http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_3.html




• DNA cloning is the best method for preparing large quantities of a particular gene or other DNA sequence.

• When the source of DNA is scanty or impure, the polymerase chain reaction (PCR) is quicker and more selective.

• This technique can quickly amplify any piece of DNA without using cells.

5. The polymerase chain reaction (PCR) clones DNA entirely in vitro


• The DNA is incubated in atest tube with special DNA polymerase, a supply of nucleotides,and short pieces ofsingle-stranded DNA as a primer.


Fig. 20.7

• PCR is very specific.• By their complementarities to sequences

bracketing the targeted sequence, the primers determine the DNA sequence that is amplified.– PCR can make many copies of a specific gene before

cloning in cells, simplifying the task of finding a clone with that gene.

– PCR is so specific and powerful that only minute amounts of DNA need be present in the starting material.

• Occasional errors during PCR replication impose limits to the number of good copies that can be made when large amounts of a gene are needed.


PCR ANIMATION• http://highered.mcgraw-hill.com/olc/dl/120

078/micro15.swf

http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

• Devised in 1985, PCR has had a major impact on biological research and technology.

• PCR has amplified DNA from a variety of sources:– fragments of ancient DNA from a 40,000-year-old

frozen wooly mammoth,– DNA from tiny amount of blood or semen found at

the scenes of violent crimes,– DNA from single embryonic cells for rapid prenatal

diagnosis of genetic disorders,– DNA of viral genes from cells infected with

difficult-to-detect viruses such as HIV.


• Gel Electrophoresis- separates linear DNA molecules, mainly on size (length of fragment) with longer fragments migrating less along the gel.


Fig. 20.8

• Restriction fragment analysis is sensitive enough to distinguish between two alleles of a gene that differ by only base pair in a restriction site.


Fig. 20.9

• For our three individuals, the results of these steps show that individual III has a different restriction pattern than individuals I or II.


Fig. 20.10

• Differences in DNA sequence on homologous chromosomes that produce different restriction fragment patterns are scattered abundantly throughout genomes, including the human genome.

• These restriction fragment length polymorphisms (RFLPs) can serve as a genetic marker for a particular location (locus) in the genome.– A given RFLP marker frequently occurs in numerous

variants in a population.


• Because RFLP markers are inherited in a Mendelian fashion, they can serve as genetic markers for making linkage maps.– The frequency with which two RFPL markers - or a

RFLP marker and a certain allele for a gene - are inherited together is a measure of the closeness of the two loci on a chromosome.


Practice

Give me the distance for:

23, 130 bp

9416 bp

6557 bp

4361 bp

2322 bp

2207 bp

564 bp

(all from lab bench)

Practice Restriction Enzyme Problem• 16. Construct a restriction map of a linear

fragment of DNA, using the following data. Your map should indicate the relative positions of the restriction sites along with distances from the ends of the molecule to the restriction sites and between restriction sites:

DNASizes of Fragments (bp)• uncut DNA 10,000• DNA cut with EcoRI 8000, 2000• DNA cut with BamHI 5000, 5000• DNA cut with EcoRI + BamHI 5000, 3000, 2000

Now you try…(expect to see one on exam!)

• DNASizes of Fragments (bp)• uncut DNA 900• DNA cut with EcoRI 700, 200• DNA cut with HindIII600, 300• DNA cut with BamHI500, 350, 50• DNA cut with EcoRI + HindIII 600, 200, 100 • DNA cut with EcoRI + BamHI 500, 200, 150, 50• DNA cut with HindIII + BamHI500, 250, 100,

50

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goal of this chapter 20: this chapter is introducing many genetic technologies that you will need to...

Documents

foreign dna slide

dna vectors

electrophoresis slide

benjamin cummings slide

complementary dna cdna

intruding dna protection

yeast cells

eukaryotic cells