Genetic Engineering

Recombinant, Cloning, Transposons, PCR’s, RFLP’s,&

Gel Electrophoresis

Intro

What is a Transposon?

• Jumping Genes• Discovered by Barbara McClintock 1940’s-50’s

• Nobel prize in 1983• Two kinds of Transposons

– Insertion Sequences• A gene which codes for the enzyme transposase. • Transposase moves genes from one DNA region to another.

May cause mutations if it transposes a gene for regulation.

– Complex transposons• Long sequences that include extra genes.

Types of Transposons

What is Junk DNA?

• DNA that does not code for a protein.• 97% of the human genome

• What is most of our DNA for?– Regulatory sequences– Introns– Polymorphic Regions: noncoding highly variable DNA

regions.– Repetitive Sequences

• Tandem repeats within a gene: Huntingtons• Tandem repeats at ends: Telomeres

Where did it come from? • One hypothesis about the junk is that these chromosomal

regions are trash heaps of defunct genes, sometimes known as pseudogenes, which have been cast aside and fragmented during evolution.

• Evidence for a related hypothesis suggests that the junk represents the accumulated DNA of failed viruses.

• Yet another hypothesis is that the junk DNA provides a reservoir of sequences from which potentially advantageous new genes can emerge. (Junk vs trash)

• As with most things in science, time and technology will tell… 

 

What are the tools of Genetic Engineering?

• Gel Electrophoresis

• DNA Probe

• Polymerase Chain Reaction

• Restriction Fragment Length Polymorphisms

• Complementary DNA

Gel Electrophosesis

• Separation of large molecules of DNA by size.

• Argose Gel in an electrical field

Gel Animation

The specifics of the Gel• DNA has a negative charge (PO4 -3). Flows from

the cathode (-) to anode (+). Hint: repels from negative.

• Can separate DNA, Proteins, or Amino Acids• Material used in Gel must by cut by restriction

enzymes to migrate through gel.• Each restriction fragment is a segment of DNA.

IE. If there is one band on the gel the DNA is uncut. If there are 2 bands the DNA is cut in 2,etc.

• The first lane represents known DNA for comparison. Running

Gel

.

So what is the use of the Gel?

• DNA fingerprinting: Who did it?

• Outbreaks: Diseases change over time (viral and bacterial morphology change)

• Paternity testing: Who’s the Daddy?

• To see if the genetically engineered product is imbedded.

AP Test Questions: Quiz Yourself

• In any electrophoresis gel, which fragment of DNA is larger?

• A) The one nearer the well

• B) The one farther from the wells

• Why?

• All of the following are true of electrophoresis except

• A) it is used to analyze only DNA

• B) the heavier the fragment the slower it moves

• C) the fragments of DNA are neg. charged and migrate to the positive pole.

• D) a buffer must cover the gel to allow a current to pass through the system.

• E) restriction enzymes cut DNA in only certain sites on the strand.

So how is DNA cut?

• Restriction Enzymes.

• Discovered in1960’s, a bacterial defense mechanism against bacteriophages.

• Examples: EcoRI(from E.coli), BamHI, HindIII

• Work by: cutting DNA at recognition sequences/sites, I.E. GAATTC

• Cuts are staggered leaving sticky ends.

• Cuts are called Restriction Fragments

Restriction Enzyme EcoRIHow is it used here?

Recombinant DNA is made by pasting sticky ends together with ligase.

So what makes your Gel Electrophoresis different from

everyone else's? (identical twins not included)

• RFLP’s: Restriction Fragment Length Polymorphisms or RFLPs– Created by DNA treated with restriction

enzymes

• The noncoding Regions of human DNA is uniquely yours = DNA Fingerprint

• Inherited through Mendalian Genetics• Uses: Paternity testing & Convictions of

Crime.

How an RFLP is analyzed

Analyze This

• .

What is recombinant DNA?

• DNA taken from two different sources and pasting them together as one.

• Two types– Natural

• Occurs during viral transduction, bacterial transformation, conjugation, and through transposons

– Artificial• Genetic engineering/ biotechnology with

restriction enzymes.

Cloning into a plasmid

Ethical Implications?

HHMI Genetic

Engineering

How can genes be cloned?

• 1.Isolate gene

• 2.Insert into Plasmid• 3.Insert into vector, I.E. competent

bacterium (a bacteria which can take up the plasmid)

• 4.Clone the gene through binary fission

• 5.Harvest successfully cloned bacteria.

• Why? Protein Production, Gene therapy, Genetic Engineering

Insulin&

Diabetes

How can a specific sequence of DNA be tagged?

• With a DNA Probe.– A radioactive label placed on a DNA fragment.– The single stranded radioactive marker binds to

its complementary sequence.

• Uses:– Gel Electrophoresis, to ID a specific sequence.– Cloning, to ensure gene uptake– To identify inherited genetic defects: Sickle cell,

Tay-Sachs, Huntington's, etc.

How can we read his DNA? or hers?

• PCR Analysis: Polymerase Chain Reaction– Automated technique which amplifies/copies

DNA. Billions of copies within hours.– Need: Nucleotides (A,T,C,& G’s), Primers, Test

tube, Taq Polymerase (Extremophile DNA Polymerase) (( heat speeds up the process))

PCR

PCR

PCR in Action

Problems with PCR

• Contamination: Need a clean room environment. No dust mites or bacteria

• Small segments of DNA: if its too big the primer will attach to the wrong thing, an incorrect DNA sequence would result.

• The nucleotide sequence must be known in order to create the correct primers.

• Did I mention contamination?

cDNA’s uses to Bioengineering

• Applications• It can be used for gene discovery or expression

analysis •  Immediate PCR Amplification of known genes•  Verification of genetic mutation•  Comparison of a specific gene between

different tissues•  Analysis of mRNA alternative splicing•  Gene cloning and target sequencing

How did we get cDNA?cDNA = complementary DNA (paging Dr. J Winters)

• Problem: Humans have introns, bacteria don’t.

• Solution: Retroviral reverse transcriptase. Makes DNA from your cells mRNA = complete coding sequence, no introns needed.

AP Test Questions: Quiz Yourself

• What is true of biotechnology techniques?

• A) PCR is used to cut DNA molecules

• B) A DNA Probe consists of a radioactive single strand of DNA

• C) Restriction Enzymes were first discovered in bacteriophage viruses.

• D) EcoR1 is the name for a DNA probe

• E) All humans contain the same RFLP’s

• Which enzyme permanently seals together DNA fragments that have complementary sticky ends?

• A) DNA Polymerase• B) Single stranded binding

protein• C) Reverse Transcriptase• D) DNA ligase• E) RNA polymerase

Ethics

• Safety: is your genetically altered food safe? Can your food alter you?

• Privacy: DNA chips, Healthcare?

• Eugenics: Insertion of genes, changing your genetics, sterilizing inferior humans, gene therapy.

GeneTherapy

And now onto your Clayton State Lab….

• Please Read the Gel.

DNA Ladder

A B

Virtual Labs

CropsMiniLab

PHS: II Electrophoresis

PHS: I Bacterial Transformation

1. Review Electrophoresis steps. Take the quiz. Paraphrase the question and correct answers. Use the semi-logarithmic paper to draw the graph with the paper given. This is part of your grade!!!!

2. Draw the steps of bacterial transformation. Take the quiz. Paraphrase the question and correct answers.

3. There are two labs here. Do both. Take notes on both. Compare and contrast the techniques. Where would you use each? What are the limitations of each? Any final thoughts or interesting points? (You must have at least one.)