DNA TechnologyDNA TechnologyChapter 20

Plasmid UsePlasmid UsePlasmids are good tools for DNA

TechnologyCan be isolated from bacterial

cellsOften carry resistance genesIsolated genes of interest can be

inserted into the plasmidHow is this insertion done?Restriction endonucleases

(enzymes)

Restriction EnzymesRestriction EnzymesWhere were restriction enzymes

first found?Bacterial cellsThey were used to protect bacteria

from intruding phage DNABacterial DNA is modified to protect

it from its own restriction enzymesRestriction enzymes often cut DNA

leaving “sticky ends”

Restriction sites are the regions on the DNA that the restriction enzyme cuts.

Why are restriction sites palindromes?

How are restriction enzymes

used in DNA technology?

Cloning Genes of InterestCloning Genes of InterestHow can a biologist make large

amounts of a gene and thereby produce lots of protein products?

Clone the genes in recombinant plasmids

Which method of bacterial genomic

alteration is exploited in

this process?

Genomic Genomic LibrariesLibraries

cDNAcDNAWhat is the problem with

inserting a human gene into a bacterial plasmid?

Introns are not spliced in prokaryotes

How can this problem be solved?Reverse Transcription of mRNA

Why is cDNA

shorter than the original eukaryotic

DNA?

ProbesProbesRadioactive probes can tag a

specific gene sequence within a mass of DNA

Probes are single stranded compliments to known sections of the DNA

Gel electrophoresisGel electrophoresisHow does gel electrophoresis

work?Uses electric charge to separate

molecules based on their sizeWhat charge does DNA have?NegativeWhich sized fragments will move

furthest through the gel?Smallest ones

Restriction Fragment Restriction Fragment AnalysisAnalysisGenetic markers are regions of

DNA that vary from person to person

Usually located on non-coding regions of the DNA

Using restriction enzymes and gel electrophoresis, DNA of different individuals can be analyzed and compared

Extract DNA and treat it with restriction enzymes

The red triangles indicate where the enzyme cuts the DNA.

Procedure:The restriction enzyme is added to the DNA being analyzed and incubated for several hours, allowing the restriction enzyme to cut at its recognition sites. The DNA is then run through a gel, which separates the DNA fragments according to size. You can then visualize the size of the DNA fragments and assess whether or not the DNA was cut by the enzyme.

Gel with an uncut and cut samples of DNA. Note that the sizes of the cut DNA fragments add up to the size of the uncut DNA.

How could you detect the differences between these 2 alleles?

Using RFLP Analysis to Using RFLP Analysis to Detect Harmful AllelesDetect Harmful AllelesHarmful, disease causing alleles

usually have identical RFLP’s within a family

Once the known RFLP’s for the normal and disease causing alleles are known family members can be tested using Southern Blot analysis

Southern Blot:Southern Blot:

Load the gel with the DNA to be tested. Markers serve as standards for determining

sizes of DNA fragments.

Separated DNA’s are denatured while still in the agarose, by soaking the gel in a basic solution

Single stranded DNA’s are transferred to a nylon membrane by blotting

A Radioactively labeled probe is added to the nylon membrane

The probe is either RNA or DNA that will compliment a specific bp sequence on the DNA

After unbound probes have been washed away only bound probes remain on the blot

VNTRVNTRA VNTR is variable numbered

tandem repeatTandem repeats are interspersed

throughout the genomeDifferent VNTR’s can be detected

using southern blot technique

One VNTR is inherited from One VNTR is inherited from each parenteach parentSouthern blot analysis usually

shows 2 different bands one inherited from each parent

How could an individual have one band for the VNTR?

He/She inherited the same sized VNTR from each parent

Three different alleles for Three different alleles for this particular VNTRthis particular VNTR

What are the different

genotypes for these 6

individuals?

Frequency of VNTR’sFrequency of VNTR’sFrequency of allele pattern at a single

VNTR has been established for specific sites within the genome

What is the probability of matching a 5 locus DNA profile, where each locus is:0.01, 0.02, 0.03, 0.06 and 0.10?

One in 27.8 million people will randomly match this profile

OJ’s profile was of 24 different loci and he matched all 24!

The odds were 1 in 10 billion

Amplification of DNAAmplification of DNAWhat enzymes would be necessary for

DNA amplification?DNA polymerase and ligaseWhat else would be necessary for the

process to work?Primers and nucleotides!How was the original DNA initially

uncoiled and unwound?HeatWhy did the heat cause difficulty with

the procedure?

Mapping the Entire Mapping the Entire GenomeGenomeGene linkage mapping uses

recombination frequencies to construct linkage maps of chromosome

Chromosome walking will identify sequential regions of the chromosome

Chromo-some

Walking

DNA Sequencing uses defective nucleotides to sequence the DNA.

In Situ HybridizationIn Situ HybridizationDenatured DNA is placed on slideradioactive single stranded probe

is used to identify complementary DNA

Used to identify genes that different species have in common

Microassays use in situ

hybridization technique to determine

which genes are actively

being expressed in

the tissue sample

Other uses for the new Other uses for the new technology….technology….

Gene Gene TherapyTherapy

Stem cells are the best

candidates for this therapy

Pharmaceutical ProductsForensicsEnvironmentAgriculturePaleontology

Determining PaternityDetermining PaternityWhich child cannot belong to this set of Which child cannot belong to this set of parents?parents?

Which lane represents the Which lane represents the father?father?

3

Rape Rape InvestigatioInvestigationnDid the suspect Did the suspect commit the commit the crime?crime?

Supplemental Lab 6ASupplemental Lab 6AIn this lab we will transform E.

coli bacterial colonies with recombinant plasmid DNA

It will be your job to distinguish between bacterial colonies that have been transformed with the recombinant plasmids

Procedure for the Procedure for the TransformationTransformation

Click here to find out more about the procedure

Predict results for your procedure!