Chapter 12 Genetic Enginneering. "In Science the credit goes to the man who convinces the...

Chapter 12Chapter 12

Genetic EnginneeringGenetic Enginneering

"In Science the credit goes to the "In Science the credit goes to the man who convinces the world, not to man who convinces the world, not to the man to whom the idea first the man to whom the idea first occurs."occurs."

– Sir Francis Darwin (1848-1925, English botanist, son of Charles – Sir Francis Darwin (1848-1925, English botanist, son of Charles Darwin), In: Eugenetics Review (April 1914, "Francis Galton") Darwin), In: Eugenetics Review (April 1914, "Francis Galton")

At the International Genetics At the International Genetics Meeting.Meeting.

An American reports:An American reports:"We crossed chickens with cows. The new breed "We crossed chickens with cows. The new breed simultaneously produces milk, meat and eggs.“simultaneously produces milk, meat and eggs.“

A Frenchman reports:A Frenchman reports:"We succeeded in cross-breeding flies and bees. "We succeeded in cross-breeding flies and bees. The hybrid flies over the trash fields and produces The hybrid flies over the trash fields and produces honey.“honey.“

A Russian reports:A Russian reports:"And we crossed a melon with cockroaches. When "And we crossed a melon with cockroaches. When you cut this melon, seeds run away by you cut this melon, seeds run away by themselves."themselves."

DNA SequencingDNA Sequencing

Genes can be cloned in Genes can be cloned in Recombinant PlasmidsRecombinant Plasmids

bb

Genes can be stored in genomic Genes can be stored in genomic LibrariesLibraries

Use of Reverse Transcriptase Use of Reverse Transcriptase

Nuc. Acid Probes are used to Nuc. Acid Probes are used to identify genes of interestidentify genes of interest

ss

Gel Electrophoresis apparatiGel Electrophoresis apparati

Bad gelsBad gels

Southern BlotsSouthern Blots

Figure 5.35 from Hart & Jones, 1998 (page Figure 5.35 from Hart & Jones, 1998 (page 207)207)

First, you run a "normal" DNA gelFirst, you run a "normal" DNA gel

(usually agarose, but it doesn't HAVE to be).(usually agarose, but it doesn't HAVE to be).

then you hybridize the DNA onto a membranethen you hybridize the DNA onto a membrane

Next, this membrane is soaked in a Next, this membrane is soaked in a mixture containingmixture containing

radioactively labelled probe, which radioactively labelled probe, which should hybridizeshould hybridize

The filter is washed and exposed to The filter is washed and exposed to radioactive film.radioactive film.

RFLPsRFLPs

restriction enzyme-generated restriction enzyme-generated fragments of different length fragments of different length

molecular markers used in creating molecular markers used in creating genetic maps of chromosomes. genetic maps of chromosomes.

relies on hybridization between a relies on hybridization between a probe and homologous DNA probe and homologous DNA segments within a genome. segments within a genome.

Polymorphism means the existence Polymorphism means the existence of many different forms in which a of many different forms in which a region of DNA can have different region of DNA can have different lengths on different homologs or in lengths on different homologs or in different individuals.different individuals.

RFLP analysisRFLP analysis

Although two individuals of the same species have almost Although two individuals of the same species have almost identical genomes, they will always differ at a few nucleotides. identical genomes, they will always differ at a few nucleotides.

Some of these differences will produce new restriction sites (or Some of these differences will produce new restriction sites (or remove them), and the banding pattern seen on a genomic remove them), and the banding pattern seen on a genomic Southern will thus be affected. Southern will thus be affected.

For any given probe (or gene), it is often possible to test different For any given probe (or gene), it is often possible to test different restriction enzymes until one which gives a pattern difference restriction enzymes until one which gives a pattern difference between two individuals is found, that is, a RFLP. between two individuals is found, that is, a RFLP.

The less related the individuals, the more divergent their DNA The less related the individuals, the more divergent their DNA sequences are and the more likely you are to find a RFLP.sequences are and the more likely you are to find a RFLP.

a semen stain left on the victim's clothing a semen stain left on the victim's clothing (EVIDENCE #1); (EVIDENCE #1); semen removed from the vagina of semen removed from the vagina of the rape victim (EVIDENCE #2); the rape victim (EVIDENCE #2);

the DNA of the victim herself (VICTIM) the DNA of the victim herself (VICTIM) to be sure that the DNA didn't come from to be sure that the DNA didn't come from her cells; her cells;

DNA from two suspectsDNA from two suspects

a set of DNA fragments of known and a set of DNA fragments of known and decreasing length (MARKER).. decreasing length (MARKER).. the cells of a previously-tested person to be the cells of a previously-tested person to be sure the probes are performing properly (CONTROL).sure the probes are performing properly (CONTROL).

thermal cycler for PCR thermal cycler for PCR

Chromosome walking is a method in Chromosome walking is a method in geneticsgenetics for identifying and for identifying and sequencingsequencing long parts of a long parts of a DNADNA strand,. strand,.

The basic technique is as follows:The basic technique is as follows: A A primerprimer that matching the beginning of the DNA that matching the beginning of the DNA

to sequence is used to synthesize a short DNA to sequence is used to synthesize a short DNA strand complementary to the unknown sequence, strand complementary to the unknown sequence, starting with the primer (see starting with the primer (see PCRPCR). ).

The new short DNA strand is sequenced using the The new short DNA strand is sequenced using the chain termination methodchain termination method. .

The end of the sequenced strand is used as a The end of the sequenced strand is used as a primer for the next part of the long DNA primer for the next part of the long DNA sequence. sequence.

Linkage MappingLinkage Mapping

Allows to tell where they are using Allows to tell where they are using recombination frequenciesrecombination frequencies

DNA SequencingDNA Sequencing

Physical Map ( order of fragments)Physical Map ( order of fragments)– Chromosome walkingChromosome walking

TransposonsTransposons

Insertion Sequences or Insertion Sequences or IS elementsIS elements simplest mobile element. simplest mobile element.

consist of a fairly short (700 - 1500 consist of a fairly short (700 - 1500 bp) DNA segment flanked by a 10 - bp) DNA segment flanked by a 10 - 40 bp inverted repeat sequence. 40 bp inverted repeat sequence.

The segment codes for the protein The segment codes for the protein ((transposasetransposase) that catalyses the ) that catalyses the transposition event:transposition event:

Composite transposons are DNA Composite transposons are DNA segments flanked by an segments flanked by an ISIS element element at either end. at either end.

In other words, instead of each In other words, instead of each ISIS element moving independently, they element moving independently, they now act in concert and move now act in concert and move together along with the intervening together along with the intervening DNA. DNA.

Practical ApplicationsPractical Applications

1. Medical Applications1. Medical Applications

– Diagnosis of DiseasesDiagnosis of Diseases

– Human Gene TherapyHuman Gene Therapy

2. Pharmaceutical Products2. Pharmaceutical Products

Vaccines, Growth hormones, insulin, Vaccines, Growth hormones, insulin, etc… etc…

3. Forensics Evidence 3. Forensics Evidence

DNA fingerprints from a murder case.  This autoradiograph shows that DNA in blood from the defendant's clothes matches the DNA fingerprint of the victim but differs from the DNA fingerprint of the defendant. This is evidence that the blood on the defendant's clothes came from the victim, not the defendant.

4. Environmental Cleanups4. Environmental Cleanups-Oil spills-Oil spills

-Mining -Mining

Bacteria being sprayed onto soil Bacteria being sprayed onto soil prepared for Bio-Remediation prepared for Bio-Remediation

5. Agricultural Applications5. Agricultural Applications

Pharm” animals.  These transgenic sheep carry a gene for a human blood protein, which they secrete In their milk. This protein inhibits an enzyme that contributes to lung damage in patients with cystic fibrosis and some other chronic respiratory diseases. Easily purified from the sheep′s milk, the protein is currently under evaluation as a treatment for cystic fibrosis.