Advanced Molecular Biological Techniques

C.S.I How do crime scene investigators (like Dexter) perform so many genetics tests when they often only find one cell at the scene? How do C.S.I’s identify suspects through DNA?

Polymerase Chain Reaction (PCR) Mullis first proposed PCR in 1987 Allows creation of more copies of DNA (20 cycles can give over a million copies!) Similar to DNA Replication PCR Animation

PCR Denaturation Temperature is increased to separate the DNA strands HEAT separates the strands Denaturation Temperature is increased to separate the DNA strands 94-96 degrees breaks hydrogen bonds between the nitrogenous base pairs

PCR Annealing DNA Primers anneal Temperature drops to 50-65 degrees Added to 3’ end of specific sequence (region of interest) Both strands of DNA are used. 5’-3’ DNA primers

Suppose this is the portion of DNA to be amplified. Cycle 1: The DNA is denatured by heating to 95oC DNA primers are included with the DNA sample. DNA primers bind to DNA when cooled to 60oC Taq polymerase along with deoxynucleotides are also part of the mixture. 5' …A T G C T T G C A A T C C G A T T C A C C G C T …3' 5' G C T T G 3' 3' A A G T G 5' 3' …T A C G A A C G T T A G G C T A A G T G G C G A… 5'

5' …A T G C T T G C A A T C C G A T T C A C C G C T …3' Cycle 1: When the mixture is warmed to 72oC, Taq polymerase produces the complementary DNA strand in the 5’ to 3’ direction. 5' …A T G C T T G C A A T C C G A T T C A C C G C T …3' 3’…T A C G A A C G T T A G G C T 3’A A G T G 5' A A G T G 5' 5' G C T T G 3’ 5' G C T T G C A A T C C G A T T C A C C G C T…3’ 3' …T A C G A A C G T T A G G C T A A G T G G C G A… 5'

PCR Extension Taq polymerase builds complimentary strands using free nucleotids Extracted from Thermus aquaticus

DNA is heated to 95oC again and denatured. Cycle 2: DNA is heated to 95oC again and denatured. Primers are annealled to DNA strands when cooled to 60oC. Taq polymerase extends the strands when warmed to 72oC. 3’ … T A C G A A C G T T A G G C T 5' …A T G C T T G C A A T C C G A T T C A C C G C T …3' C A A T C C G A T T C A C 3’ 3’…T A C G A A C G T T A G G C T A A G T G 5' 5' G C T T G C A A T C C G A T T C A C C G C T…3’ 3' …T A C G A A C G T T A G G C T A A G T G G C G A… 5' 3’ C G A A C G T T A G G C T C A A T C C G A T T C A C C G C T… 3’ Notice the appearance of strands of DNA of different length. These are referred to as “variable length fragments. 5' G C T T G A A G T G 5' 5' G C T T G A A G T G 5'

PCR Exponential Amplification Process is repeated and region of interest is amplified exponentially. Minutes per cycle

“Restriction Fragment Length Polymorphism” (RFLP) the difference in DNA fragment lengths (when cut by restriction enzymes) between individuals Allows scientists to match DNA from a crime scene to a suspect, obtain diagnosis of genetic diseases, and to determine paternity/maternity RFLP animation

The Thermal Cycler The repetitive nature of heating and cooling lends itself to automation. Table top thermal cyclers are no larger than a bread-baking machine. The thermal cycler will take a mixture consisting of the original DNA sample, Taq polymerase, forward and reverse primers and free deoxynucleotides, all in a 0.5 mL tube and produce a billion copies in just a few hours.

The Thermal Cycler Note the display: the thermal cycler is programmed for 20 cycles DNA is denatured at 94°C for 30 s primers anneal at 55°C for 1 minute and Taq polymerase is given 3 minutes at 68°C to extend the primers and produce the complementary strand.

“Restriction Fragment Length Polymorphism” (RFLP) Differences in a DNA sequence between individuals Organisms of the same species carry the same genes but differ in their respective alleles. Genomes of individuals of the same species are polymorphic (unless identical twins) POLYMORPHISM - any detectable difference in DNA

“Restriction Fragment Length Polymorphism” (RFLP) Polymorphism in exons. To identify individuals with specific mutations. Possible polymorphism in introns (VNTRs) Variable number tandem repeats TAGTAGTAGTAGTAG…. Unique to individuals

“Restriction Fragment Length Polymorphism” (RFLP) DNA digested with restriction endonuclease(s) Cut DNA at specific palindromic points Left with sticky ends

“Restriction Fragment Length Polymorphism” (RFLP) Gel Electrophoresis Fragments separate based on size and charge

“Restriction Fragment Length Polymorphism” (RFLP) DNA denatured into single strands Southern blotting. Banding patterns transferred from gel to nylon membrane using electric current

“Restriction Fragment Length Polymorphism” (RFLP) Membrane soaked in a solution containing radioactive complimentary nucleotide probes. DNA sequence tagged Base pairing occurs between target DNA and probe (known as hybridization)

“Restriction Fragment Length Polymorphism” (RFLP) Autoradiogram produced Nylon membrane placed against X-ray film for 2-3 weeks. Probes burn image on to the film.

RFLP Analysis Southern Blot

Homework

APPLICATIONS Gene therapy PCR applications RFLP applications

Gene therapy Refers to any method for treating genetic diseases that involves altering the DNA sequence Inserting genes Deleting genes Altering expression of genes Can act on either the germ line cells (results will be heritable), or the somatic cells

Insertion Inserting genes can be accomplished by introducing vectors into the host cell Viral transfection Direct injection of DNA Insertion can occur at a random location: risk of altering existing host gene

Altering expression Use an antisense oligonucleotide “oligonucleotide” – A short nucleic acid (RNA) strand “antisense” – Complementary to a functional mRNA Introduce short antisense RNA strands Complementary base-pairing with mRNA will occur  prevents translation Use to de-activate specific mRNA’s associated with disease

Effectiveness of antisense gene therapy has so far been limited Clinical trials: HIV/AIDS Cancer High cholesterol Ebola hemorrhagic fever Pain management in cancer patients Read section 6.4 to find out more about this

Applications of PCR Useful when only a small amount of DNA is available Archaeological samples “degraded DNA" Forensic investigations DNA evidence may be limited Medical diagnosis e.g., HIV virus. Amplification allows detection before immune system symptoms are widespread

Applications of RFLP Genetic screening Some genetic diseases are associated with particular RFLP banding patterns e.g., Sickle cell anemia – base pair substitution occurs within restriction site for DdeI Similar techniques can be used to screen for known genetic mutations Digest DNA and hybridize probes that are complementary to mutations Requires blood sample or another biological sample Prenatal screening: use amniotic fluid

Forensic investigations and Paternity testing DNA Fingerprinting Forensic investigations and Paternity testing Location of restriction sites is unique to individuals Digest genomic DNA with several RE’s Banding pattern should be particular to each individual Compare suspect banding patterns with those from crime scene samples or from child Forensics: Looking for 100% concordance Paternity: Looking for 50% concordance

Side note: DNA profiles today... RFLP is time-consuming and requires large amounts of DNA PCR-based techniques are actually used today for generating DNA profiles Why do you think RFLP-based DNA fingerprinting is an unattractive alternative for forensic investigations?

VNTR’s (microsatellites) are the markers of choice The copy number will vary between individuals PCR is used to selectively amplify certain VNTR loci so the number of repeats can be determined Separation occurs by electrophoresis, but within a narrow glass capillary tube instead of a slab of gel

Who da babydaddy??? IS THE ALLEGED FATHER THE BABYDADDY?? Assign “names” to RFLP variants Determine genotypes of sources Compare: Child should share one RFLP variant with father, one with mother As a rule, Child/AF mix should not have more than three bands Source Genotype Mother B/E Child B/D Alleged father (A.F.) A/C Child/A.F. mix A/B/C/D A B C D E IS THE ALLEGED FATHER THE BABYDADDY??  NO! Follow link for more detail

IS THE ALLEGED FATHER THE BABYDADDY??  YES! Source Genotype Mother B/D Child C/D Alleged father (A.F.) A/C Child/A.F. mix A/C/D A B C D IS THE ALLEGED FATHER THE BABYDADDY??  YES!

To catch a killer... Two suspects Two samples recovered from scene Victim shares no bands with either suspect Crime Scene 2 sample: Victim is the source Crime Scene 1 sample: Whodunnit?

PCR Animations http://www.dnalc.org/ddnalc/resources/animations.html http://www.amnh.org/learn/pd/genetics/pcr/interactive.html