Unit # 8 - DNA 1 “The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and.

Unit # 8 - DNAUnit # 8 - DNA 11

“The capacity toblunder slightly is thereal marvel of DNA.Without this specialattribute, we would stillbe anaerobic bacteria andthere would be no music.”

—Lewis Thomas, Physician, author


DNA AnalysisDNA Analysis

Students will learn: That DNA is a long-chain polymer found in nucleated cells, which contain genetic information. That DNA can be used to identify or clear potential suspects in crimes. How DNA is extracted and characterized. How to apply the concepts of RFLP, PCR, and STRs to characterize DNA. The role that statistics plays in determining the probability that two people would have the same sequence in a

fragment of DNA.


DNA AnalysisDNA Analysis

Students will be able toStudents will be able to:: Explain that DNA is a long molecule, tightly packed Explain that DNA is a long molecule, tightly packed

in the form of a chromosome with genetic material in the form of a chromosome with genetic material wrapped around it. wrapped around it.

Isolate and extract DNA from cells. Isolate and extract DNA from cells. Describe the function and purpose of a restriction Describe the function and purpose of a restriction

enzyme. enzyme. Calculate probabilities of identity using STR. Calculate probabilities of identity using STR.


Historical InformationHistorical Information James Watson and Francis CrickJames Watson and Francis Crick——1953 1953

discovered the configuration of the DNA moleculediscovered the configuration of the DNA molecule

Ray WhiteRay White——1980 describes first polymorphic 1980 describes first polymorphic RFLP markerRFLP marker

Alec JeffreysAlec Jeffreys——1985 isolated DNA markers and 1985 isolated DNA markers and called them DNA fingerprintscalled them DNA fingerprints

Kary MullisKary Mullis——1985 developed PCR testing1985 developed PCR testing

19881988——FBI starts DNA caseworkFBI starts DNA casework

19911991——first STR paperfirst STR paper

19981998——FBI launches CODIS databaseFBI launches CODIS database


People of Historical People of Historical SignificanceSignificance

James Watson, Francis Crick, and Maurice Wilkins James Watson, Francis Crick, and Maurice Wilkins jointly received the Nobel Prize in 1962 for their jointly received the Nobel Prize in 1962 for their determination of the structure of DNA. What is determination of the structure of DNA. What is interesting about this fact is that Rosalind Franklin had interesting about this fact is that Rosalind Franklin had as much to do with the discovery as the other three as much to do with the discovery as the other three gentlemen with her work with X-ray crystallography. gentlemen with her work with X-ray crystallography. She died of cancer and could not be honored for her She died of cancer and could not be honored for her work. Find out more at Chemical Achievers: work. Find out more at Chemical Achievers:

www.chemheritage.org/EducationalServices/chemach/ppb/cwwf.htmlwww.chemheritage.org/EducationalServices/chemach/ppb/cwwf.html


General DNA InformationGeneral DNA Information Double helixDouble helix——two coiled DNA strandstwo coiled DNA strands Composed of nucleotidesComposed of nucleotides——units containing a sugar units containing a sugar

molecule (deoxyribose), phosphate group and a molecule (deoxyribose), phosphate group and a nitrogen-containing basenitrogen-containing base

In humans, the order of these bases is 99.9% the In humans, the order of these bases is 99.9% the same.same.

Four basesFour bases AdenineAdenine CytosineCytosine GuanineGuanine ThymineThymine

Bases always pair A to T and G to CBases always pair A to T and G to C


Where Is DNA Found?Where Is DNA Found? Genes are portions of DNA that code for Genes are portions of DNA that code for

specific proteins specific proteins DNA is found in all nucleated body cellsDNA is found in all nucleated body cells

——white blood cells, semen, saliva, urine, white blood cells, semen, saliva, urine, hair root, teeth, bone, tissuehair root, teeth, bone, tissue

Most abundant in buccal (cheek) cellsMost abundant in buccal (cheek) cells Red blood cells have no nuclei; and Red blood cells have no nuclei; and

therefore, no nuclear DNAtherefore, no nuclear DNA DNA obtained from blood comes from DNA obtained from blood comes from

white blood cellswhite blood cells


DNA TypingDNA Typing

DNA typing is a method in which DNA DNA typing is a method in which DNA is converted into a series of bands that is converted into a series of bands that ultimately distinguish each individual. ultimately distinguish each individual. Only one-tenth of a single percent of Only one-tenth of a single percent of DNA (about 3 million bases) differs DNA (about 3 million bases) differs from one person to the next. Scientists from one person to the next. Scientists use these regions to generate a DNA use these regions to generate a DNA profile of an individual.profile of an individual.


Non-Coding RegionsNon-Coding Regions

3 percent of the human DNA sequences 3 percent of the human DNA sequences code for proteinscode for proteins

97 percent is non-coding and is repetitive; 97 percent is non-coding and is repetitive; repeating the same sequence over and repeating the same sequence over and overover

50 percent of the human genome has 50 percent of the human genome has interspersed repetitive sequencesinterspersed repetitive sequences


Uses of DNA ProfilingUses of DNA Profiling

To identify potential suspectsTo identify potential suspects To exonerate individualsTo exonerate individuals To identify crime and casualty victimsTo identify crime and casualty victims To establish paternityTo establish paternity To match organ donorsTo match organ donors


DNA TYPINGDNA TYPING“Fingerprinting”“Fingerprinting”

RFLPRFLP——Restriction Fragment Length Restriction Fragment Length PolymorphismPolymorphism

PCRPCR——Polymerase Chain ReactionPolymerase Chain Reaction

STRSTR——Short Tandem RepeatsShort Tandem Repeats


RFLP—Restriction Fragment RFLP—Restriction Fragment Length PolymorphismsLength Polymorphisms

Restriction enzymes are used to cut DNA into Restriction enzymes are used to cut DNA into smaller fragments that can then be separated smaller fragments that can then be separated and characterized for identificationand characterized for identification IsolateIsolate——separate DNA from the cellseparate DNA from the cell

CutCut——using restriction enzymes to make shorter using restriction enzymes to make shorter base strandsbase strands

SortSort——by size using electrophoresisby size using electrophoresis

AnalyzeAnalyze——the specific alleles for identificationthe specific alleles for identification


PCR—PolymerasePCR—PolymeraseChain ReactionChain Reaction

PCR is a technique used for making PCR is a technique used for making copies of a defined segment of a DNA copies of a defined segment of a DNA molecule. This can be valuable when molecule. This can be valuable when the amount of evidence is minimal. the amount of evidence is minimal. Millions of copies of DNA can be made Millions of copies of DNA can be made from a single speck of blood.from a single speck of blood.


PCR—Polymerase Chain PCR—Polymerase Chain Reaction ProcedureReaction Procedure

Heat the DNA strands, causing the Heat the DNA strands, causing the strands to separate (unzip).strands to separate (unzip).

Cool the mixture and add a primer, a short Cool the mixture and add a primer, a short sequence of base pairs that will add to its sequence of base pairs that will add to its complementary sequence on the DNA complementary sequence on the DNA strand.strand.

Finally, add a DNA polymerase and a Finally, add a DNA polymerase and a mixture of free nucleotides to the mixture of free nucleotides to the separated strands. Heat again to around separated strands. Heat again to around 7575°° C for the completion.C for the completion.


PCRPCR——PolymerasePolymeraseChain ReactionChain Reaction

The outcome is a doubling of the The outcome is a doubling of the number of DNA strands. Heating, number of DNA strands. Heating, cooling, and strand rebuilding is cooling, and strand rebuilding is repeated typically 25 to 30 times, repeated typically 25 to 30 times, yielding more than one million copies yielding more than one million copies of the original DNA molecule. Each of the original DNA molecule. Each cycle takes less than two minutes cycle takes less than two minutes from start to finish.from start to finish.


Advantages of PCRAdvantages of PCR Minute amounts of DNA may be used for amplification.Minute amounts of DNA may be used for amplification. DNA degraded to fragments only a few hundred base DNA degraded to fragments only a few hundred base

pairs in length can serve as effective templates for pairs in length can serve as effective templates for amplification.amplification.

Large numbers of copies of specific DNA sequences Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR can be amplified simultaneously with multiplex PCR reactions.reactions.

Commercial kits are now available for easy PCR Commercial kits are now available for easy PCR reaction setup and amplification.reaction setup and amplification.

Contaminant DNA, such as fungal and bacterial Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific sources, will not amplify because human-specific primers are used. However, human contamination can primers are used. However, human contamination can be a problem.be a problem.


ElectrophoresisElectrophoresis A technique used to separate DNA A technique used to separate DNA

fragments.fragments.

An electrical current is moved through a An electrical current is moved through a gel substance causing molecules to sort gel substance causing molecules to sort by size.by size.

The smaller, lighter molecules will move The smaller, lighter molecules will move the furthest on the gel. the furthest on the gel.

After developing, the fragments can be After developing, the fragments can be visualized for characterization.visualized for characterization.


ElectrophoresisElectrophoresis

Pipette the DNA.



Load DNA into the Load DNA into the gel wells.gel wells.



Run the gel. Run the gel.

Observe and Observe and compare bands of compare bands of DNA.DNA.


Short Tandem Repeats (STR)Short Tandem Repeats (STR)STR is another method of DNA typing. STR’s STR is another method of DNA typing. STR’s are locations (loci) on the chromosome that are locations (loci) on the chromosome that contain short sequences of 2 to 5 bases that contain short sequences of 2 to 5 bases that repeat themselves in the DNA molecule. The repeat themselves in the DNA molecule. The advantages of this method are that it provides advantages of this method are that it provides greater discrimination, requires less time, a greater discrimination, requires less time, a smaller sample size, and the DNA is less smaller sample size, and the DNA is less susceptible to degradation.susceptible to degradation.



Short Tandem Repeats Short Tandem Repeats (STR) Procedure(STR) Procedure

Extract the gene TH01 from the sample. (TH01 Extract the gene TH01 from the sample. (TH01 has seven human variants with a repeating has seven human variants with a repeating sequence of A-A-T-G)sequence of A-A-T-G)

Amplify the sample by means of PCRAmplify the sample by means of PCR

Separate by electrophoresisSeparate by electrophoresis

Examine the distance the STR migrates to Examine the distance the STR migrates to determine the number of times TH01 repeatsdetermine the number of times TH01 repeats


Short Tandem Repeats (STR)Short Tandem Repeats (STR)

Each person has two STR types for TH01Each person has two STR types for TH01——one inherited from each parent.one inherited from each parent.

By continuing the process with additional By continuing the process with additional STRs from other genes, you can narrow STRs from other genes, you can narrow down the probability of DNA belonging to down the probability of DNA belonging to only one person.only one person.


Short Tandem Repeats (STR)Short Tandem Repeats (STR)

STR typing is visualized by peaks shown on a STR typing is visualized by peaks shown on a graph. Each represents the size of the DNA graph. Each represents the size of the DNA fragment.fragment.

The possible alleles are numbered for each The possible alleles are numbered for each loci.loci.


Profiler Plus Allelic Ladders

D3S1358 FGAVWA

AMEL D8S1179 D21S11 D18S51

D5S818 D13S317D7S820


COfiler Allelic Ladders

D3S1358

AMEL

D7S820

D16S539

TH01TPOX CSF1PO


STR ExampleSTR Example



Determining ProbabilityDetermining Probability

Databases have been established that Databases have been established that determine how often a particular allele on determine how often a particular allele on a loci appears in a given population. By a loci appears in a given population. By increasing the number of alleles on increasing the number of alleles on different loci the probability of having two different loci the probability of having two people with the exact combination people with the exact combination becomes miniscule.becomes miniscule.


DNA InteractiveDNA Interactive

The website below has a STR animation The website below has a STR animation demonstration. Click on human demonstration. Click on human identification, profiling and then on the identification, profiling and then on the third circle called Today’s DNA Profiling to third circle called Today’s DNA Profiling to see the demonstration.see the demonstration.

http://www.dnai.org/d/index.htmlhttp://www.dnai.org/d/index.html


Three Possible OutcomesThree Possible Outcomes MatchMatch——The DNA profile appears the The DNA profile appears the

same. Lab will determine the frequency.same. Lab will determine the frequency. ExclusionExclusion——The genotype comparison The genotype comparison

shows profile differences that can only be shows profile differences that can only be explained by the two samples originating explained by the two samples originating from different sources.from different sources.

InconclusiveInconclusive——The data does not The data does not support a conclusion as to whether the support a conclusion as to whether the profiles match.profiles match.


Types of DNATypes of DNANuclearNuclear found in the nucleusfound in the nucleus constitutes 23 pairs of constitutes 23 pairs of

chromosomes inherited chromosomes inherited from both parentsfrom both parents

each cell contains only each cell contains only one nucleione nuclei

MitochondrialMitochondrial found in the cytoplasmfound in the cytoplasm is inherited only from is inherited only from

mother mother each cell contains each cell contains

hundreds to thousands hundreds to thousands of mitochondriaof mitochondria

can be found in skeletal can be found in skeletal remainsremains

Nuclear DNA is present in the head of the sperm. Mitochondrial DNA is present in the tail. At conception, the head of the sperm enters the egg and unites with the nucleus. The tail falls off, losing the father’s mitochondrial DNA.


Mitochondrial DNAMitochondrial DNA

Analysis of mDNA is moreAnalysis of mDNA is more:: rigorousrigorous time consuming time consuming costly than nucleic testing of DNAcostly than nucleic testing of DNA

mDNA is constructed in a circular or loopmDNA is constructed in a circular or loop 37 genes are involved in mitochondrial 37 genes are involved in mitochondrial

energy generationenergy generation Is used when nuclear DNA typing is not Is used when nuclear DNA typing is not

possiblepossible


FBI’s CODIS DNA DatabaseFBI’s CODIS DNA Database

CoCombined mbined DDNA NA IIndex ndex SSystemystem Used for linking serial crimes and Used for linking serial crimes and

unsolved cases with repeat offendersunsolved cases with repeat offenders Launched October 1998Launched October 1998 Links all 50 statesLinks all 50 states Requires >4 RFLP markers and/or 13 Requires >4 RFLP markers and/or 13

core STR markerscore STR markers


The FutureThe Future

Greater automation of the DNA typing processGreater automation of the DNA typing process

Use of SNP’sUse of SNP’s——single nucleotide polymorphism single nucleotide polymorphism which measures a one nucleotide change or which measures a one nucleotide change or difference from one individual to another. More sites difference from one individual to another. More sites are needed to differentiate between individuals (30 are needed to differentiate between individuals (30 to 50 SNPs to attain the frequencies of the 13 STR to 50 SNPs to attain the frequencies of the 13 STR loci), but it can be done with robots and automation.loci), but it can be done with robots and automation.


People in the NewsPeople in the News

Sir Alec JeffreysSir Alec Jeffreys is credited with DNA profiling using is credited with DNA profiling using RFLP. In September of 1984 after years of work, he RFLP. In September of 1984 after years of work, he saw his first series of blots on an X-ray. The saw his first series of blots on an X-ray. The technique was first used in forensics, when in 1985 technique was first used in forensics, when in 1985 he was asked by police to confirm the rape he was asked by police to confirm the rape confession of 17 year old Richard Buckland, who was confession of 17 year old Richard Buckland, who was denying a rape of another young woman. The DNA denying a rape of another young woman. The DNA from Buckland and the DNA taken from the victims from Buckland and the DNA taken from the victims eliminated him as a suspect. Jefferys then used eliminated him as a suspect. Jefferys then used samples from other suspects to later convict Colin samples from other suspects to later convict Colin Pitchfork whose DNA did match.Pitchfork whose DNA did match.

Unit # 8 - DNA 1 “The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and.

Documents

portions of dna

dna profile

structure of dna

fragment of dna

extract dna

single percent of dna

dna moleculeray white1980

dnauses of dna profilingto