Section 6.3

Advanced Techniques in Molecular Biology

OutlineTechniques

1. Polymerase chain reaction2. Restriction fragment length polymorphism + Southern

blotting3. DNA sequencing

Applications1. Gene therapy2. 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 sampleThe 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

Steps1. De-naturation of DNA (separation of strands)2. Priming of DNA3. Elongation of new DNA strand

Separation of strandsIn DNA replication, which enzyme performs this

function?

In PCR, heat (94°C - 96°C) is used to denature the strandsBreaks the H-bonds

Priming of DNA templateDNA primers are engineered in the lab

designed to be complementary to the template

Primers are annealed to the 3’ ends of the template strandsTwo different primers – the “forward” and “reverse”

primers

Temperature is reduced to allow annealing (50-65°C)

ElongationDNA Polymerase adds deoxyribonucleotides to

the 3’ end of the primer

Recall enzymes are denatured by heatDNA Pol III is denatured at 37°C

Heat-resistant polymerase is used: Taq polymeraseThermus aquaticus, hot springs bacterium

Cycle repeats over and over to produce many copies:

Video:http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

CyclingTarget strand is not completely isolated after one roundOne 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 allelesNon-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 polymorphicIt will be different between individuals

The differences in restriction fragment length can be used to individualize DNA samples

Steps1. DNA is digested with restriction enzymes & denatured2. The digested sample is separated by gel electrophoresis3. Radioactive probes specific to the target sequence are

hybridized to the sequences4. 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 DNAThe sample will appear to the eye as a continuous

smear of bandsNeed to “highlight” the target sequence

Southern blottingThe separated DNA needs to be transferred out of the gel

in order to hybridize with the probe

Procedure:Nylon membrane is placed on the gelElectric current is applied:

(+) behind nylon; (-) behind gelNegatively-charged DNA will transfer to the nylon

Video:http://highered.mcgraw-hill.com/olc/dl/120078/bio_g.swf

Detecting the target sequenceNylon membrane is immersed in a

solution with the radioactive probes

Allow probes to hybridize by complementary base pairing

Nylon is placed on X-ray filmExposure of film will occur where the

radioactive probes are locatedAn autoradiogram is the pattern of bands

on the X-ray film

DNA SequencingSanger dideoxy method

Based on the process of DNA replicationUtilizes DNA synthesis reactions to determine sequence

of bases in synthesized strand

Sequencing reactionsRequires 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 templateDNA polymerasefree nucleotides

“regular” deoxyribonucleotides (dNTPs), as well as a deoxyribonucleotide analogue (ddNTPs)

Deoxyribonucleotide analogueCalled a dideoxy analogueLike 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

ProcessIn each reaction tube, allow synthesis to occurDNA polymerase will add on free nucleotides to the

end of the primerChain elongation will occurWhenever 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 occurHowever, 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’s will greatly outnumber ddNTP’s.

Reduces the probability of a ddNTP being incorporated whenever the complementary base is encountered.

End result:Each reaction tube will contain fragments of different

lengthsFragment 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

Gel electrophoresisto separate fragments in each sample

Lanes:Sequencing reaction for GSequencing reaction for ASequencing reaction for CSequencing reaction for T

Allow the gel to runSouthern blotDetect fragments (expose X-ray film)

Primers were radioactive

Recall shorter fragments will migrate fartherFragments will differ by only one base pair

Reading the sequenceRead backwards from the positive electrode to

determine the sequence

APPLICATIONS•Gene therapy•PCR applications•RFLP applications

Gene therapyRefers to any method for treating genetic diseases

that involves altering the DNA sequenceInserting genesDeleting genesAltering expression of genes

Can act on either the germ line cells (results will be heritable), or the somatic cells

InsertionInserting genes can be accomplished by introducing

vectors into the host cellViral transfectionDirect injection of DNA

Insertion can occur at a random location: risk of altering existing host gene

Altering expressionUse an antisense oligonucleotide

“oligonucleotide” – A short nucleic acid (RNA) strand “antisense” – Complementary to a functional mRNA

Introduce short antisense RNA strandsComplementary base-pairing with mRNA will occur

prevents translationUse to de-activate specific mRNA’s associated with disease

Effectiveness of antisense gene therapy has so far been limited

Clinical trials:HIV/AIDSCancerHigh cholesterolEbola hemorrhagic feverPain management in cancer patients

Read section 6.4 to find out more about this

Applications of PCRUseful when only a small

amount of DNA is availableArchaeological samples

“degraded DNA"Forensic investigations

DNA evidence may be limited

Medical diagnosise.g., HIV virus. Amplification allows detection before

immune system symptoms are widespread

Applications of RFLPGenetic screeningSome genetic diseases are associated with particular RFLP

banding patternse.g., Sickle cell anemia – base pair substitution occurs within

restriction site for DdeI

Similar techniques can be used to screen for known genetic mutationsDigest DNA and hybridize probes that are complementary to

mutationsRequires blood sample or another biological samplePrenatal screening: use amniotic fluid

DNA Fingerprinting

Forensic investigations and Paternity testingLocation of restriction sites is unique to individualsDigest 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 childForensics: Looking for 100% concordancePaternity: Looking for 50% concordance

Side note: DNA profiles today...RFLP is time-consuming and requires large amounts of DNAPCR-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??? 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

BC

D

EIS THE ALLEGED FATHER THE BABYDADDY?? NO! Follow link for more detail

A

BC

D

Source Genotype

Mother B/D

Child C/D

Alleged father (A.F.)

A/C

Child/A.F. mix A/C/D

IS THE ALLEGED FATHER THE BABYDADDY?? YES!

To catch a killer...Two suspectsTwo samples recovered from sceneVictim shares no bands with either

suspect

Crime Scene 2 sample:Victim is the source

Crime Scene 1 sample:Whodunnit?

Homework