DNA evidence is a frozen moment in time pdf

PCR: Polymerase Chain Reaction• A method of in vitro cloning • Allows amplification of specific DNA molecules fragments in vitro through cycles of enzymatic DNA synthesis • The most pop

DNA evidence is a frozen moment

in time

Image by collaborator Carl Kriigel, US Army Criminal Investigation Lab

DNA is convicting the guilty and freeing

the inncocentClarence Harrison, 2004

of the courtroom a free man on Tuesday Just last week, Hampikian reviewed new DNA test results and discovered Harrison’s DNA doesn't match the evidence saved from the scene of a rape and robbery in 1986 …

How good is DNA at exonerating?

Crime labs report about 25% of

do not match primary suspect (FBI, GBI, Virginia, Connecticut, Justice Department)

Hampikian group mitochondrial projects

Where Is All This DNA Coming From?

• DNA is found in all body cells (except mature red blood cells)

• We leave a little bit of DNA everywhere we go

• Most forensic sources of DNA are body fluids, or transferred cells

DNA Use in Forensic Cases

• Most are rape cases (>2 out of 3)

• Looking for match between evidence and suspect

• Mixtures must be resolved

• DNA can be degraded (bacteria, fungi, sunlight, heat)

• Inhibitors to diagnostic test can be present (heme, dyes…)

• Scientists need a quick and easy way to produce DNA in sufficient quantities for their studies and generate labeled DNA molecules to visualize and study specific molecules within cells

Challenges

Modified from www.bioforensics.com

PCR (Polymerase Chain Reaction)

GV: TS Lê Quang Nguyên

DNA in the Cell

Target Region for PCR

What is PCR?

• PCR is an exponentially progressing synthesis of the

defined target DNA sequences in vitro

• “Polymerase” because the only enzyme used in this reaction is DNA polymerase

• “Chain” because the products of the first reaction become substrates of the following one, and so on

• It was invented in 1983 by Dr Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993

Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 1985 Dec 20;230(4732):1350-4

S aiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlic h HA, Arnheim N.

Cetus Corporation, Department of Human Genetics, Emeryville, CA 94608.

Development/Invention of PCR Technique

1993 Nobel Prize in Chemistry

PCR: Polymerase Chain Reaction

• A method of in vitro cloning

• Allows amplification of specific DNA molecules (fragments) in

vitro through cycles of enzymatic DNA synthesis

• The most popular and widely used technique in all fields of biological studies probably

PCR Amplifies a Specific DNA Seq

• PCR can be used to target a specific DNA subsequence in a much larger DNA sequence (e.g., a single 1000bp gene from the human genome, which is 3 × 109bp)

• PCR allows exponential amplification of a DNA sequence

– Each PCR cycle theoretically doubles the amount

of DNA

– During PCR, an existing DNA molecule is used as

a template to synthesize a new DNA strand.

– Through repeated rounds of DNA synthesis, large

quantities of DNA are produced.

• Robustness: will even work on degraded DNA or fixed DNA

• Simplicity: require eppendorf, thermocycler, and ingredients for PCR

• These features make PCR extremely useful in basic research and commercial applications, including genetic identity testing, forensics,

industrial quality control and quick in vitro diagnostics (virus,

bacteria,etc.).

Disadvantages of PCR

• Need for Target DNA sequence information

– To construct primers you need to know your target

• Short size limit for product

– There is an upper limit to the size of DNA

The “Reaction” Components

1) Target DNA - contains the sequence to be amplified

2) Pair of Primers - oligonucleotides that define the sequence

to be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

reaction

5) Mg ++ ions - cofactor of the enzyme

6) Buffer solution – maintains pH and ionic strength of the

reaction solution suitable for the activity of the enzyme

What is Taq polymerase?

 Most proteins denature at extreme pH or high temperatures

 Human DNA polymerase would denature at 94C New polymerase would have to be added at each elongation step

aquaticus; a bacterium that lives in hot springs.

 Many of its enzymes (including DNAP I) will not denature

at high temperatures

Thermus aquaticus, a thermophilic bacteria discovered in

1969 in hot spring of Yellowstone National park It can

tolerate high temperature The DNA polymerase (Taq polymerase) was isolated.

The PCR Process - PCR Primers

• Primers define the DNA sequence to be amplified— they give the PCR specificity.

• Primers bind (anneal) to the DNA template and act

as starting points since DNA polymerases cannot initiate DNA synthesis without a primer.

• The distance between the two primers determines the length of the newly synthesized DNA molecules.

One PCR cycle consists of a DNA denaturation step, a primer annealing step and a primer extension step.

• DNA Denaturation: Expose the DNA template to high temperatures to separate

the two DNA strands and allow access by DNA polymerase and PCR primers.

• Primer Annealing: Lower the temperature to allow primers to anneal to their

complementary sequence.

• Primer Extension: Adjust the temperature for optimal thermostable DNA

polymerase activity to extend primers.

PCR uses a thermostable DNA polymerase so that the DNA polymerase is not heat-inactivated during the DNA denaturation

step Taq DNA polymerase is the most commonly used DNA

polymerase for PCR.

The PCR Process

The PCR Process - Mechanism of DNA

Synthesis

• DNA polymerase extends the primer by sequentially adding a single dNTP (dATP, dGTP, dCTP or dTTP) that is complementary to the existing DNA strand

• The sequence of the newly synthesized strand is complementary to that of the template strand

• The dNTP is added to the 3´ end of the growing DNA strand,

so DNA synthesis occurs in the 5´ to 3´ direction

The PCR Process - Mechanism of DNA

Synthesis

?

Thermal Cycling Programs

A typical thermal cycling program is:

• Initial DNA denaturation at 95o C for 2

minutes

• 20–35 PCR cycles:

• Denaturation at 95o C for 30 seconds to 1

minute

• Annealing at 42–65o C for 1 minute

• Extension at 68–74o C for 1–2 minutes

• Final extension at 68–74o C for 5–10

minutes

• Soak at 4o C

• Thermal cyclers have a heat-conducting block to modulate reaction temperature

• Thermal cyclers are programmed to maintain the appropriate temperature for the required length of time for each step of the PCR cycle.

• Reaction tubes are placed inside the thermal cycler, which heats and cools the heat block to achieve the necessary temperature

The PCR Process - Instrumentation

Materials

Number of cycles in PCR

• n cycles  2n PCR products

• < 40 cycles, to avoid:

• - degradation and exhausting of ingredients

• - by-products which inhibit the reaction

• - copies bind to each other

PCR reagent is the limiting factor!!

PCR Optimization

Many PCR parameters might need to be optimized to increase yield, sensitivity of detection or amplification specificity These parameters include:

Magnesium Concentration

The most important factors to optimize

• The optimal Mg2+ concentration depends upon the primers, template, DNA polymerase, dNTP concentration and other factors

– Some reactions amplify equally well at a number

of Mg 2+ concentrations, but some reactions only amplify well at a very specific Mg 2+ concentration

• When first time using a set of PCR primers: titrate magnesium in 0.5 or 1.0mM increments to determine the

optimal concentration.

Primer Annealing Temperature

• PCR primers must anneal to the DNA template at the chosen

• When performing multiplex PCR, where multiple DNA targets are amplified in a single PCR, all sets of PCR primers must have similar annealing temperatures

– Primers with intramolecular complementarity

can form secondary structure within the same primer molecule.

– Intermolecular complementarity allows a primer

molecule to anneal to another primer molecule rather than the template

• Software packages exist to design primers

• Primer Size: too small may bind to more than one site in the genome Too large take a longer time to hybridize and would slow down the PCR cycle (not > 30 bp)

• Annealing temperature is most important

• Too low = non-specific binding

• Too high = primer will not bind

•Ideal annealing temperature can be mathematically estimated

It should be just 1-2 C below Tm

DNA Quality

DNA should be intact and free of contaminants that inhibit amplification.

• Contaminants can be heme from blood, humic acid from soil and melanin from hair

• Contaminants can be introduced during the purification process such as Phenol, ethanol, sodium dodecyl sulfate (SDS) and other detergents, and salts

DNA Quantity

DNA quantity

• More template is not necessarily better.

– Too much template can cause nonspecific amplification.

– Too little template will result in little or no PCR product.

• The optimal amount of template will depend on the size of the DNA molecule.

•TÁI TỔ HỢP SẢN PHẨM PCR = GENE

 Gắn 2 trình tự DNA khác nhau thông qua PCR (vd: gắn promoter và gene)

 Gắn các điểm nhận biết của enzyme cắt giới

hạn vào primers  dòng hoá sản phẩm PCR vào vector

RECOMBINANT PCR

GẮN CÁC ĐIỂM NHẬN BIẾT CỦA ENZYME CẮT

GIỚI HẠN VÀO PRIMERS

DÒNG HOÁ NHỜ T-A

Taq polymerase leaves an “A”

overhang

• Taq is the thermostable DNA polymerase from Thermus

aquaticus we used for PCR.

• When Taq synthesizes a new strand, it always puts an extra

“A” at the end

• This can be useful, but note: other polymerases do not do

this, they leave “blunt” ends Only Taq polymerase

leaves ‘A’ overhangs ‘Blunt’ end vectors do not work with Taq, we need a ‘T’ overhang.

Site specific mutagenesis

PCR mutagensis

RT-PCR ( R EVERSE T RANSCRIPTION)

• Reverse transcriptases are RNA-dependent DNA polymerases, which use an RNA template to make a DNA copy (cDNA) This cDNA can be amplified using PCR

DNA

DNA DNA

DNA DNA

DNA

DNA Reverse

transcription

PCR

DNA DNA

RT-PCR Components

Typical components of an RT-PCR include:

• Reverse transcriptase: the enzyme that synthesizes the cDNA copy of

the RNA target.

• Reverse transcription primer: a single short DNA molecule that acts

as starting points for the reverse transcriptase, since reverse transcriptases cannot initiate DNA synthesis without a primer.

• Deoxynucleotide triphosphates (dNTPs): the building blocks for the

newly synthesized cDNA.

• Reaction buffer: a chemical solution that provides the optimal

environmental conditions

• Magnesium: a necessary cofactor for reverse transcriptase activity.

• All of the necessary PCR components for the PCR portion of PCR.

RT-RT-PCR

• LABELING DNA IN HYBRIDIZATION

Labeling DNA with tags for use as tools (probes) to visualize complementary DNA or RNA molecules

•Radioactive labels: probes will darken an X-ray film

•Fluorescent labels (nonradioactive)

• Fluors will absorb light energy of a specific wavelength (the excitation wavelength) and emit light at a different wavelength (emission wavelength)

• The emitted light is detected by specialized instruments such as fluorometers

DNA and RNA Detection

• PCR can detect foreign DNA sequences in a biological sample.

– Example: Hospitals often use PCR to detect bacteria and

viruses and help diagnose illnesses.

• PCR can detect specific DNA sequences to characterize an organism.

– Example: The multidrug resistance (MDR) gene confers

resistance to antibiotics that are commonly used to treat bacterial infections PCR using primers specific for the MDR gene will identify strains of bacteria that express MDR and are resistant to common antibiotics.

• RT-PCR can detect specific RNA sequences within a sample.

– Example: Retroviruses have an RNA genome Retroviral

RNA can be detected by RT-PCR to diagnose retroviral infections.

Detection Of PathogensMolecular Identification:

Detection Of Pathogens

Sensitivity of detection of PCR-amplified M tuberculosis DNA (Kaul et al.1994)

Molecular Identification:

Genotyping and DNA-Based

Identification

• Cellular (genomic) DNA contains regions of variable sequences that differ between strains or even individual organisms

• Variable regions are amplified by multiplex PCR, and when the resulting DNA fragments are separated by size, the resulting pattern acts like a unique barcode to identify a strain or individual

– For human identification, these variable regions

often include short tandem repeats (STRs) and single-nucleotide polymorphisms (SNPs).

– STRs and SNPs are useful in DNA-based forensic

investigations, missing persons investigations and paternity disputes.

Short Tandem Repeats

Genotyping by STR markersMolecular Identification:

A Homologous Chromosome Pair

• One from mom

• One from dad

• What is the difference?

Image: greghampikian@bosiestate.edu

13 CODIS Core STR Loci

CSF1PO D5S818

D21S11

TH01 TPOX

D13S317

D7S820

D8S1179 D3S1358

FGA

VWA

AME L

AME L

www.cstl.nist.gov/biotech/strbase/ppt/4

Forensic STR (Single Tandem Repeat) analysis looks at

the length of 13-16 areas of DNA

DNA-Based Human

Identification

The police collect a hair from

a crime scene and submit it for STR analysis (sample #1) Five suspicious people were observed near the crime scene shortly after the crime was committed The police collect DNA from these five people and submit it for STR analysis (samples #2–6).

Do any of these five DNA samples match the DNA from the hair collected at the cime scene?

MULTIPLEX PCR

DNA and RNA Quantitation

of a DNA sequence such as a gene within a genome or the number of organisms present in a sample (e.g., determining viral load)

messenger RNA (mRNA) produced in a cell

– As gene expression within a cell is activated or

repressed, the level of corresponding mRNA increases or decreases, respectively

– Quantitating mRNA levels by RT-PCR can tell us

which genes are being up- or downregulated under certain conditions, providing insight into gene function.

REAL TIME PCR

Theo dõi hàm lượng sản phẩm PCR mục tiêu theo thời gian

thật (real time) của phản ứng

- Chất hoá học phát huỳnh quang có khả năng gắn vào mọi

phân tử DNA mạch đôi

- Các probe có đánh dấu bằng chất phát huỳnh quang để lai

đặc hiệu với sản phẩm PCR đích:

* TaqMan probes

* FRET probes using the LightCycler

* Molecular Beacons

- Amplicon được đo sau mỗi chu kì PCR

- Hàm lượng sản phẩm khuếch đại được theo dõi thông qua

tín hiệu phát huỳnh quang

CHU KÌ NGƯỠNG (Threshold cycle-Ct)

• - Ct: là chu kì mà tại đó tính hiệu huỳnh quang mẫu vượt trên tín hiệu huỳnh quang nền (vd

quencher/Taqman probe) của phản ứng PCR 

thiết bị cảm ứng ghi nhận

• - Nguyên tắc định lượng của real time PCR dựa vào đường cong chuẩn (standard curve) được xây dựng từ các Ct của các mẫu có nồng độ acid nucleic

(amplicon) biết trước