Advanced Techniques in Molecular Biology

1. Advanced Techniques in Molecular Biology Section 6.3

2. Outline Techniques • Polymerase chain reaction • Restriction fragment length polymorphism + Southern blotting • DNA sequencing Applications • Gene therapy • Other applications

3. ADVANCED TECHNIQUES Polymerase chain reaction Restriction length polymorphism & Southern blotting DNA sequencing

4. Polymerase Chain Reaction (PCR) • Technique for making many copies of DNA from a small sample • The sample DNA is said to be “amplified” • Contrast with cloning in a plasmid: direct method of isolating and replicating a desired DNA sequence

5. Technique is modeled after DNA replication • Uses some of the same machinery

6. Steps • De-naturation of DNA (separation of strands) • Priming of DNA • Elongation of new DNA strand

8. Separation of strands • In DNA replication, which enzyme performs this function? • In PCR, heat (94°C - 96°C) is used to denature the strands • Breaks the H-bonds

9. Priming of DNA template • DNA primers are engineered in the lab • designed to be complementary to the template • Primers are annealed to the 3’ ends of the template strands • Two different primers – the “forward” and “reverse” primers • Temperature is reduced to allow annealing (50-65°C)

10. Elongation • DNA Polymerase adds deoxyribonucleotides to the 3’ end of the primer • Recall enzymes are denatured by heat • DNA Pol III is denatured at 37°C • Heat-resistant polymerase is used: Taq polymerase • Thermus aquaticus, hot springs bacterium

11. Cycle repeats over and over to produce many copies: Video: http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

12. Cycling • Target strand is not completely isolated after one round • One cycle produces variable-length strands • After the second cycle, the target strand is isolated. • Constant-length strands are produced. • Third cycle onwards: The number of target strands increases exponentially

14. Restriction Fragment Length Polymorphism (RFLP) • Polymorphism: Any difference in DNA sequence that can be detected between individuals. Can be in either a coding, or a non-coding region. Individuals within a species are polymorphic. • Coding polymorphisms are alleles • Non-coding polymorphisms: includes variable number tandem repeats (microsatellites), restriction fragment length

15. RFLP analysis: The principle • Restriction fragment: A region that is flanked by restriction sites. The sequence in between the sites is the “target” sequence. • The length of the target sequence is polymorphic • It will be different between individuals • The differences in restriction fragment length can be used to individualize DNA samples

16. Steps • DNA is digested with restriction enzymes & denatured • The digested sample is separated by gel electrophoresis • Radioactive probes specific to the target sequence are hybridized to the sequences • Distinctive banding pattern will be detected, depending on the location of restriction sites Video: http://highered.mcgraw-hill.com/olc/dl/120078/bio20.swf

17. Why are probes needed? • Genomic DNA is an extremely large source of DNA • The sample will appear to the eye as a continuous smear of bands • Need to “highlight” the target sequence

18. Southern blotting • The separated DNA needs to be transferred out of the gel in order to hybridize with the probe Procedure: • Nylon membrane is placed on the gel • Electric current is applied: (+) behind nylon; (-) behind gel • Negatively-charged DNA will transfer to the nylon Video: http://highered.mcgraw-hill.com/olc/dl/120078/bio_g.swf

19. Detecting the target sequence • Nylon membrane is immersed in a solution with the radioactive probes • Allow probes to hybridize by complementary base pairing • Nylon is placed on X-ray film • Exposure of film will occur where the radioactive probes are located • An autoradiogram is the pattern of bands on the X-ray film

20. DNA Sequencing • Sanger dideoxy method • Based on the process of DNA replication • Utilizes DNA synthesis reactions to determine sequence of bases in synthesized strand

21. Sequencing reactions • Requires four separate synthesis reactions • In each of the four reaction tubes, place the following components: • DNA to be sequenced (denatured first) • a short, radioactively-labelled primer , complementary to end of template • DNA polymerase • free nucleotides • “regular” deoxyribonucleotides (dNTPs), as well as a deoxyribonucleotide analogue (ddNTPs)

22. Deoxyribonucleotide analogue • Called a dideoxy analogue • Like a deoxyribonucleotide, except does not have a –OH group on the 3’ carbon • Free nucleotides cannot be added onto the 3’ end of a dideoxy analogue

23. Sequencing setup: • Each reaction tube will locate a different nucleotide (base) where it is incorporated into the new strand • One tube each for G, A, T, and C • "G" tube: all four dNTP's, ddGTP and DNA polymerase • "A" tube: all four dNTP's, ddATP and DNA polymerase • "T" tube: all four dNTP's, ddTTP and DNA polymerase • "C" tube: all four dNTP's, ddCTP and DNA polymerase

24. Process • In each reaction tube, allow synthesis to occur • DNA polymerase will add on free nucleotides to the end of the primer • Chain elongation will occur • Whenever a ddNTP is incorporated into the chain, synthesis will STOP

25. e.g., sequence to be elucidated: 5’-TTACGTACGTAA-3’ • If a ddATP is incorporated instead of dATP, termination of synthesis will occur • However, sometimes a regular dATP will be incorporated, allowing several possible fragments: 5’-TTA-3’ 5’-TTACGTA-3’ 5’-TTACGTACGTA-3’ 5’-TTACGTACGTAA-3’

26. This same process occurs in each of the four reaction tubes, but for different bases

27. To ensure that productive chain elongation occurs, dNTP’s will greatly outnumber ddNTP’s. • Reduces the probability of a ddNTP being incorporated whenever the complementary base is encountered.

28. End result: • Each reaction tube will contain fragments of different lengths • Fragment length depends on where a ddNTP was added to the chain • Through random incorporation of nucleotides, theoretically a fragment should exist that corresponds to every location of that base in the sequence

29. Analysis • Gel electrophoresisto separate fragments in each sample • Lanes: • Sequencing reaction for G • Sequencing reaction for A • Sequencing reaction for C • Sequencing reaction for T

30. Allow the gel to run • Southern blot • Detect fragments (expose X-ray film) • Primers were radioactive • Recall shorter fragments will migrate farther • Fragments will differ by only one base pair

31. Reading the sequence • Read backwards from the positive electrode to determine the sequence

32. APPLICATIONS Gene therapy PCR applications RFLP applications

33. 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

34. 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

35. Altering expression • Use an antisenseoligonucleotide • “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

36. 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

37. Applications of PCR • Useful when only a smallamount 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

38. 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

39. 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

40. 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?

41. 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

42. Who da 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 A B C D E IS THE ALLEGED FATHER THE BABYDADDY??  NO! Follow link for more detail

43. A IS THE ALLEGED FATHER THE BABYDADDY?? YES! B C D

44. 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?

45. Homework