Advanced Techniques in Molecular Biology

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Advanced Techniques in Molecular Biology. Section 6.3. Outline. Techniques Polymerase chain reaction Restriction fragment length polymorphism + Southern blotting DNA sequencing Applications Gene therapy Other applications. ADVANCED TECHNIQUES. Polymerase chain reaction
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Advanced Techniques in Molecular Biology Section 6.3 Outline Techniques
  • Polymerase chain reaction
  • Restriction fragment length polymorphism + Southern blotting
  • DNA sequencing Applications
  • Gene therapy
  • Other applications
  • ADVANCED TECHNIQUES Polymerase chain reaction Restriction length polymorphism & Southern blotting DNA sequencing 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
  • Technique is modeled after DNA replication
  • Uses some of the same machinery
  • Steps
  • De-naturation of DNA (separation of strands)
  • Priming of DNA
  • Elongation of new DNA strand
  • 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
  • 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)
  • 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
  • Cycle repeats over and over to produce many copies: Video: http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • DNA Sequencing
  • Sanger dideoxy method
  • Based on the process of DNA replication
  • Utilizes DNA synthesis reactions to determine sequence of bases in synthesized strand
  • 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)
  • 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
  • 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
  • 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
  • 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’
  • This same process occurs in each of the four reaction tubes, but for different bases
  • To ensure that productive chain elongation occurs, dNTP but for different bases’s will greatly outnumber ddNTP’s.
  • Reduces the probability of a ddNTP being incorporated whenever the complementary base is encountered.
  • End result: but for different bases
  • 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
  • Analysis but for different bases
  • Gel electrophoresisto separate fragments in each sample
  • Lanes:
  • Sequencing reaction for G
  • Sequencing reaction for A
  • Sequencing reaction for C
  • Sequencing reaction for T
  • Allow the gel to run but for different bases
  • 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
  • Reading the sequence but for different bases
  • Read backwards from the positive electrode to determine the sequence
  • APPLICATIONS but for different bases Gene therapy PCR applications RFLP applications Gene therapy but for different bases
  • 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 but for different bases
  • 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 but for different bases
  • 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
  • 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 limited
  • 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
  • Applications of RFLP limited 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
  • DNA Fingerprinting limited
  • 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... limited
  • 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 limited’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??? limited
  • 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 A limited IS THE ALLEGED FATHER THE BABYDADDY?? YES! B C D To catch a killer... limited
  • 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?
  • Homework limited
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