JS 196e: Scientific Basis of DNA Typing continued and Methods used to Study DNA

I. Pre class activitiesa. Quizb. Assignment and announcements

II. Learning ObjectivesDefinition and Inheritance reviewIntroduction to DNA- continued

a. DNA structureb. Be able to draw DNA structure – Base

Pairing AT, GCc. Understand DNA replication

Summary of Introduction to DNA

Overview of Methods used in Forensic DNA typing ScreeningExtractionQuantification

Assignments and Announcements

• Friday 9 Feb: Guest lecture by Dr. Chris Chen of CCNY on nanotechnology- 1 point extra credit for attending and writing a 500 word summary

• Required assignments for 12 Feb– Work on Laboratory Notebooks

– Study for a quiz

– Read Butler C3/InmanC2

– Review : Special collection guidelines for Biological Evidence- http://www.cacnews.org/wordfiles/DNA%20SampleHandling.doc

Where’s Daddy?PCR product size (bp)

11 14

11

12 14

8 14

12

128

Inheritance Review

Dad is homozygous (A, A) and mom is heterozygous (A, a).

In your teams, draw the Punnett Sqaure that demonstrates the inheritance of these alleles.

What percentage of their children will be heterozygous?

DNA StructureWhat is it?

Bases (AGCT) form the stairs of the ladder, are faithfully paired and exhibit differences.

PS-A : T-SP PS-G : C-SP PS-A : T-SP PS-G : C-S P

 Sugars (S) and phosphates (P) form the sides of the ladder (identical for all DNA).

Bases (AGCT) form the stairs of the ladder, are faithfully paired by hydrogen bonds and exhibit differences. A : T and G : C

A = T

G C

T = A

A = T

C G

T

C

C

A

G

G

T

A

G C

T = A

T = A

C G

A = T

A = TG C

5’

3’

3’

5’ 3’

3’ 5’denaturedstrands

hybridizedstrands

Hydrogen bonds

C G C

G

G C

Phosphate-sugar backbone

DNA Structure

• Primary genetic material is composed of two complementary strands

• Form a double helix or twisted ladder

• Sides are sugar phosphate and the steps are base pairs

• Four Bases- 2 Purines – Adenine and Guanine and 2 Pyrimidines- Cytosine and Thymine.

DNA StructureNucleotides are the building blocks themselves

composed of PBS

Nucleotides-PBSPhosphate (negative charge) Base (AGCT-Asian Guys Can Teach)Sugar (deoxyribose-5C)

Phosphate-SugarsConnected by phosphodiester linkages

DNA Structure2 Complimentary,

Antiparallel Strands held together by Base Pairs- H Bonds

A:T held with 2 H Bonds G:C held with 3 H Bonds.

Replication of DNA is Semi ConservativeOne old and one new

http://dir.niehs.nih.gov/dirlmg/repl.html

Enzymes of Replication

DNA is replicated or copied in our cells. When completed, the new double strands consist of one old template and one newly made strand- This is called semi conservative replication.

There are many enzymes that are required. They include unwinding (helicases, gyrases), priming (primases), copying (DNA polymerases) and touch up enzymes (DNA ligases).

DNA replication is quite accurate in our cells. The error rate is approximately 10-9

O

Base(A, T, C, or G)

HO

1’

3’ 2’

4’

5’CH2OP

H

HH

H

O

O-

HO

O

Base(A, T, C, or G)

HOH

1’

3’ 2’

4’

5’CH2OP

H

HH

H

O-

HO

P-P-

OH

dNTP

3-OH’

5-P’

Template = Old

Primer5-P’

3-OH’

DNA polymerase

Mg++

PCR: repeated rounds of DNA Replication •5 required ingredients (components)- primer, template, Mg, dntps, DNA polymerase- PTMDD-(please to make DNA doubled)

•DNA Polymerase catalyzes the template directed (A-T, G-C), incorporation of dNTPs (PP is released) forming a 3’-5’ phosphodiester linkage

• Direction of synthesis 5’3’ using primer 3’OH to attach incoming nucleotide

DNA St. Patricks Day Salute to the Molecule of HeredityFrom Biology 110- UNC 1993 Steve Lee

The molecular structure today Is heredity’s DNAWith nucleotides completely comprised of a sugar and phosphate and base

The bases you see are so keenThey include thymine and adenineCytosine and one more with guanine can store all the info with rungs in between

The sides of the ladder you know,are sugar and phosphate which showthat Franklin was rightdouble helix is tightten base pairs per turn in a row

Adenine and thymine can base pairForming two hydrogen bonds for one stairCytosine and guaninepair with three in betweenand are equal in size when compared

DNA strands are just not the sameOne is coding and one is called lame (anticoding)They are opposite in direction and thisis called antiparallel in name

Complimentary nature of strandslets replication proceed just as plannedwith A paring to Tand G pairing to Cthe fidelity is precise and quite grand

Steps in Forensic DNA typing(Figure 6.1 Rudin and Inman 2001)Evaluation- Is it there?

1. Start with biological sample

2. Screen- blood? Semen? Saliva, human?Extraction- Get and clean DNA3. Open cells Get DNA

4. Methods to get DNA and purify DNA

Quantify- Determine quality and quantity?

5. Quantify- How good and how much did you get?

Type to determine and compare alleles 6. RFLP vs PCR

7. Determine alleles and compare DNA typesOr alleles present in samples and references

Interpretation of Results

Review: DNA is organized inside the cell nucleus and mitochondria

DNA Extraction

After screening tests are performed, a spot of the material containing the biological sample is cut and placed into a tube.

In one type of extraction method (organic), heat and chemicals are added, and protein is removed. Then the pure DNA is recovered by filtration in which the non-DNA material goes through a sieve.(analogous to a collection of your pasta in a colander)

Different DNA Extraction Methods

The organic method generally yields the highest quantity and quality DNA. The main disadvantages are that it is tedious with lots of steps and utilizes corrosive chemicals.The Chelex method is used when the sample contains very few cells and the reduced number of handling steps is the primary advantage. The main disadvantage is that it is yields crude DNA that is not as pureFTA paper is used to collect reference samples. It can be stored at room temperature, requires minimal handling and no quantification is required.

Differential Extraction MethodFor Sexual Assault Evidence

• Isolation of DNA from mixtures of cells in sexual assault evidence

• Based on differences in cell membranes

– Spermatozoa membranes have special cross links (sulphur-sulphur bonds)

– These membranes are quite resistant to opening.

– Vaginal epithelial cells do not contain these membranes and are more easily broken open

Sperm and v cellmixture

Lysis- openv cell extractFemale DNA

Female DNA

Male DNA

Femalecell

Spermatozoa

Lysis- open

sperm extract

Male DNA

Quantification of DNA

• Following extraction, the next step is to determine the quantity of the DNA

• DNA typing methods RFLP and PCR require different amounts and different quality of DNA.

• RFLP typically required 50ng. PCR typically requires less than 0.5ng to 1 ng: 100 times less!

Quantification of DNA using Gel Electrophoresis

• Total DNA can be quantified by running the samples in a gel.

• Typically, gels are made up of agarose (a carbohydrate from seaweed).

• Known DNA quantities are included• Samples are then subject to an electric

current and is called electrophoresis.• DNA is negatively charged and will

migrate toward the positive electrode… • Comparisons of the results are done

visually or with computer software to determine the amount of DNA in the unknown sample.

(-)

(+)

wells

IntactDNA

Degraded

DNA

L K K – u u u

Direction of DNA fragment movementSmaller fragments move faster and are foundNear the bottom of the gel

Slot Blot quantification : DNA-DNA Hybridization

DNA is where it’s AT

• DNA samples may contain non human DNA

• In order to quantify the amount of human DNA in a sample, a human specific test is required

• One such test is DNA-DNA hybridization using a human specific probe: D17Z1

Slot blot hybridization• Like in yield gels, known amounts of DNA (human) are

included

• DNA hybridization of D17Z1 will occur only if the sample contains human DNA

• Detection of the hybridized fragments is done using an enzyme linked assay- yielding light or color

Real Time PCR

Quantitative PCR- QPCRhttp://pathmicro.med.sc.edu/RTPCR/rt-pcr.ppt

• Real-time QPCR has several advantages over the other methods in that it is extremely accurate and sensitive over a broad dynamic range, and it occurs in a closed-tube system, reducing the potential for carryover contamination.

• Using this technique, a forensic biologist can monitor and quantify the accumulation of PCR products during log phase amplification. (Heid et al., 1996).

• Several RT PCR human specific assays are now available that target autosomal, Alu repeats, Y chromosome and mtDNA (Andréasson et al. 2002, Nicklas et al. 2003, Green et al. 2005, Andréasson et al. 2006, Horsman et al. 2006).

• The assays may be performed on single targets or in multiplexes (Timken et al. 2005, Walker et al. 2005, Nicklas et al. 2006).

• Recently, the detection of degraded vs intact human DNA and PCR inhibitors has been reported (Swango et al. 2006).

RNA based quantification methods• Different genetic expression patterns (mRNAs) exist in different

tissue types. • Body fluid identification has been reported based on their mRNA

profiles (Juusola and Ballantyne 2003 and 2005, Nussbaumer et al. 2006)

• In addition, the age of a bloodstain was reported using analysis of mRNA: rRNA ratios (Anderson et al. 2005). This information may be useful in establishing the time of the crime.

• Advantages of the mRNA-based approach, versus the conventional biochemical tests, include greater specificity, simultaneous and semi-automatic analysis, rapid detection, decreased sample consumption and compatibility with DNA extraction methodologies.

• The quantification of the amounts of the mRNA species relative to housekeeping genes is a critical aspect of the assays (Juusola and Ballantyne 2003).

Comparison of Methods used for DNA Quantification

Method Ease Cost Sensitivity Result

UV Spectrophotometry +++++ + ++ Total DNA

Yield Gel electrophoresis +++ + + Int vs deg DNA

Slot Blot ++ ++ +++ Human DNA

Yield Gel blot + ++ +++ Int. vs. deg human DNA Pico-green microtitre plate ++++ ++ ++++ Total DNA

Alu Quant +++ +++ ++++ Human DNA

Real time PCR assays +++ +++ +++++ Human DNA

Real time PCR assays +++ +++ +++++ Int.vs. deg.Human DNA

Summary 1 (from last week)•Why study DNA

– Law enforcement, evolution, agricultural, and human applications-medical diagnostics

•DNA Biology and Genetics– DNA is contained in cells –the basic unit of life– Found in nuclei, mitochondria and chloroplasts– Organized in chromosomes. Located at positions called loci and come in different

forms or alleles. – Homozygous if the same, heterozygous if different– Alleles segregate independently and assort randomly when on different

chromosomes. Random assortment is desired for forensic DNA loci.

• DNA Function and Structure– DeoxyriboNucleic Acid : blueprints of life

Replication, Information storage and mutation RIM– Central Dogma

DNA------->RNA------>proteintranscription translation

Summary 2• DNA Structure and Function continued:

– Bases are Adenine, Guanine, Cytosine and Thymine- Asian Guys Can Teach: AGCT– Base pairing is A to T and G to C- DNA is where its AT– Sequence of Bases Store information- Like the sequence of numbers in a Phone Number– Sides of the ladder are Sugar-Phosphate backbones– Nucleotides are the building blocks (dNTPs) themselves made of phosphate base and sugar= PBS-

The only station Sierra and Gabriel can watch– DNA base pairs- DNA velcro (David Letterman

• DNA Replication – Semi-conservative- Half republican (old) /half democrat (new)– Template directed with base pairing (AT, GC)– 5 required ingredients of PCR - primer, template, Mg, dntps, DNA

polymerase (PTMDD)

Summary 3

• Steps in forensic DNA typing are – Evaluation, Extraction, Quantification, Typing and Interpretation– 1) Evaluation- Is it there? Screen- blood? Semen?

Saliva, human?– 2) Extraction- Get and clean DNA

• Open cells -Get DNA• Organic, Chelex and FTA extractions

• Quantify- Determine quality and quantity?– How good and how much did you get?– Yield Gels, Slot Blots, Real time PCR assays

Case #2: Can you extract and type DNA from fingerprint powder that has been used to develop latent prints? If so, design an experiment with + and – controls to support your hypothesis.

Quantification References• Anderson S, Howard B, Hobbs GR, Bishop CP. (2005) A method for determining the age of a bloodstain. Forensic Sci Int. 2005 Feb 10;148(1):37-45. Links • Andreasson H, Gyllensten U, Allen M. (2002) Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis. Biotechniques. 2002 Aug;33(2):402-4, 407-11. • Andreasson H, Nilsson M, Budowle B, Lundberg H, Allen M. (2006) Nuclear and mitochondrial DNA quantification of various forensic materials. Forensic Sci Int. 2006 Jan 18;• Budowle B, Hudlow WR, Lee SB, Klevan L. (2001) Using a CCD camera imaging system as a recording device to quantify human DNA by slot blot hybridization. Biotechniques. 2001 Mar;30(3):680-5. • Butler, J (2005) Forensic DNA Typing: Biology and Technology Behind STR Markers ISBN: 0-12-147952-8, 688pp. Academic Press.• Fox JC, Cave CA, Schumm JW. (2003) Development, characterization, and validation of a sensitive primate-specific quantification assay for forensic analysis. Biotechniques. 2003 Feb;34(2):314-8, 320,

322. Links • Green RL, Roinestad IC, Boland C, Hennessy LK. (2005) Developmental validation of the quantifiler real-time PCR kits for the quantification of human nuclear DNA samples. J Forensic Sci. 2005

Jul;50(4):809-25. • Heid CA, Stevens J, Livak KJ, Williams PM. (1996) Real time quantitative PCR. Genome Res. 1996 Oct;6(10):986-94. • Horsman KM, Hickey JA, Cotton RW, Landers JP, Maddox LO. (2006) Development of a human-specific real-time PCR assay for the simultaneous quantitation of total genomic and male DNA. J

Forensic Sci. 2006 Jul;51(4):758-65. • Jones (2005)- Forensic Science Handbook Saferstein editor Forensic Science Handbook, Volume II, 1/e Richard Saferstein, Bill Bliss, Arlington, VA ©1988 / ISBN: 0133268772• Juusola J, Ballantyne J. (2003) Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Forensic Sci Int. 2003 Aug 12;135(2):85-96. • Juusola J, Ballantyne J. (2005) Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int. 2005 Aug 11;152(1):1-12. • Kline, M.C., Duewer, D.L., Redman, J.W., Butler, J.M. (2003) NIST mixed stain study 3: DNA quantitation accuracy and its influence on short tandem repeat multiplex signal intensity. Anal. Chem. 75:

2463-2469.• Kline, M.C., Duewer, D.L., Redman, J.W., Butler, J.M. (2005) Results from the NIST 2004 DNA Quantitation Study. J. Forensic Sci., 50(3): 571-578.• Mandrekar MN, Erickson AM, Kopp K, Krenke BE, Mandrekar PV, Nelson R, Peterson K, Shultz J, Tereba A, Westphal N. (2001) Development of a human DNA quantitation system. Croat Med J. 2001

Jun;42(3):336-9. • Nicklas JA, Buel E. (2003) Development of an Alu-based, QSY 7-labeled primer PCR method for quantitation of human DNA in forensic samples, Journal of Forensic Sciences, Vol 48, No 2, 282-291,

2003. • Nicklas JA, Buel E, (2003) Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples, Journal of Forensic Science, Vol 48, No 5, 936-944, 2003. • Nicklas JA, Buel E, (2003) Quantitation of DNA in Forensic Samples, Analytical and Bioanalytical Chemistry, Vol 376, No. 8, 1160-1167, 2003.• Nicklas JA, Buel E. (2006) Simultaneous determination of total human and male DNA using a duplex real-time PCR assay. J Forensic Sci. 2006 Sep;51(5):1005-15• Nussbaumer C, Gharehbaghi-Schnell E, Korschineck I. (2006) Messenger RNA profiling: a novel method for body fluid identification by real-time PCR. Forensic Sci Int. 2006 Mar 10;157(2-3):181-6.

Epub 2005 Nov 9. • Swango, KL, MD.Timken, M.Date-Chong, MR Buoncristiani (2006) A quantitative PCR assay for the assessment of DNA degradation in forensic samples. Forensic Science International 158:14-26.• Timken MD, Swango KL, Orrego C, Buoncristiani MR (2005) A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: Implications for

quantifying DNA in degraded samples. J Forensic Sci. Sep;50(5):1044-1060.• Walker JA, Hedges DJ, Perodeau BP, Landry KE, Stoilova N, Laborde ME, Shewale J, Sinha SK, Batzer MA. (2005) Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear,

mitochondrial, and male Y-chromosome DNA: application in human identification. Anal Biochem. 2005 Feb 1;337(1):89-97. • Walsh, PS, J. Varlaro, and R. Reynolds (1992) A rapid chemiluminescent method for quanititation of human DNA Nucl. Acids Res. 1992 20: 5061-5065.• Waye JS, Presley LA, Budowle B, Shutler GG, Fourney RM. (1989) A simple and sensitive method for quantifying human genomic DNA in forensic specimen extracts. Biotechniques. 1989 Sep;7(8):852-

5. • Singer VL, Jones LJ, Yue ST, Haugland RP. (1997) Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.Anal Biochem.

1997 Jul 1;249(2):228-38. Links