DNA Technology Chapter 20

Plasmid Use Plasmids are good tools for DNA Technology Can be isolated from bacterial cells Often carry resistance genes Isolated genes of interest can be inserted into the plasmid How is this insertion done? Restriction endonucleases (enzymes)

Restriction Enzymes Where were restriction enzymes first found? Bacterial cells They were used to protect bacteria from intruding phage DNA Bacterial DNA is modified to protect it from its own restriction enzymes Restriction 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 Interest How 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 Libraries

cDNA What 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?

Probes Radioactive probes can tag a specific gene sequence within a mass of DNA Probes are single stranded compliments to known sections of the DNA

Gel electrophoresis How does gel electrophoresis work? Uses electric charge to separate molecules based on their size What charge does DNA have? Negative Which sized fragments will move furthest through the gel? Smallest ones

Restriction Fragment Analysis Genetic 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 Detect Harmful Alleles Harmful, 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: 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

VNTR A VNTR is variable numbered tandem repeat Tandem repeats are interspersed throughout the genome Different VNTR’s can be detected using southern blot technique

One VNTR is inherited from each parent Southern 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 this particular VNTR What are the different genotypes for these 6 individuals?

Frequency of VNTR’s Frequency 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 DNA What enzymes would be necessary for DNA amplification? DNA polymerase and ligase What else would be necessary for the process to work? Primers and nucleotides! How was the original DNA initially uncoiled and unwound? Heat Why did the heat cause difficulty with the procedure?

Mapping the Entire Genome Gene 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 Hybridization Denatured DNA is placed on slide radioactive 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 technology….

Stem cells are the best candidates for this therapy Gene Therapy Stem cells are the best candidates for this therapy

Pharmaceutical Products Forensics Environment Agriculture Paleontology

Determining Paternity Which child cannot belong to this set of parents?

Which lane represents the father? 3

Rape Investigation Did the suspect commit the crime?

Supplemental Lab 6A In 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 Transformation Click here to find out more about the procedure Predict results for your procedure!