PCR

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 inncocent
Clarence Harrison, 2004
Local DNA expert helps overturn Georgia conviction
08:56 AM PDT on Wednesday, September 1, 2004 Adam Atchison / KTVB
.Thanks in part to the efforts of Hampikian and his team, Georgia resident Clarence Harrison walked out 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 samples sent by law enforcement do not match primary suspect (FBI, GBI, Virginia, Connecticut, Justice Department)
Hampikian group mitochondrial projects
Other activities:
Basque mitochondrial heritage study of 95 unrelated families
Murder of an Alaskan Native Chief
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
Blood
Semen
Saliva
Urine
Hair
Teeth
Bone
Tissue
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
chromosome
cell nucleus
Double stranded DNA molecule
Individual nucleotides
www.cstl.nist.gov/biotech/strbase/ppt/4
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.
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich 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.
1989 - 219
1990 – 496 1998/10 - >73,000
1991 – 711 1999/4 - >81,000
1992 – 906 2000/10 – 121,305
1993 –1030 2001/2 – 125,563
1994 – 857 2002/3 – 149,572
1995 – 823 2003/2 – 170,841
1996 – 796 2004/2/23-195,193
1997 – 732 2004/2/26-195,265
2006/3/22 - 255,788
2006/4/18 – 257,737
2007/3/9 – 283,607
2007/4/11 - 286,486
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.


Advantages of PCR
PCR is one of the most useful techniques in laboratories today due to its speed and sensitivity.
Traditional techniques to amplify DNA require days or week. PCR can be performed in as little as 1-3 hours.
Many biochemical analyses require the input of significant amounts and certain purity of biological material; PCR requires as little as one DNA molecule.
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 synthesized by PCR
Infidelity of replication
Because the PCR polymerases are heat stable they tend not to have the 3’->5’ exonuclease activity. (Taq polymerase generates 1 error in 10000 nucleotides)
Impurity
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.
4) Thermostable DNA Polymerase - enzyme that catalyzes the 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.
Taq polymerase is the DNA polymerase I for Thermus 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 95oC for 2 minutes
20–35 PCR cycles:
Denaturation at 95oC for 30 seconds to 1 minute
Annealing at 42–65oC for 1 minute
Extension at 68–74oC for 1–2 minutes
Final extension at 68–74oC for 5–10 minutes
Soak at 4oC
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!!
Copies of DNA=2N
PCR Optimization
Many PCR parameters might need to be optimized to increase yield, sensitivity of detection or amplification specificity. These parameters include:
Magnesium concentration
Primer annealing temperature
PCR primer design
DNA quality
DNA quantity
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 Mg2+ concentrations, but some reactions only amplify well at a very specific Mg2+ 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 annealing temperature.
The optimal annealing temperature depends on the length and nucleotide composition of the PCR primers
The optimal annealing temperature is often within 5oC of the melting temperature (Tm) of the PCR primer
Tm is defined as the temperature at which 50% of complementary DNA molecules will be annealed (i.e., double-stranded).
When performing multiplex PCR, where multiple DNA targets are amplified in a single PCR, all sets of PCR primers must have similar annealing temperatures.
PCR Primer Design
Ideally all primers used in a PCR will have similar melting temperatures (45–70oC) and GC content (~50%).
Primers should have little intramolecular and intermolecular secondary structure, which can interfere with primer annealing to the template.
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.
Tm = (4 x [G+C]) + (2 x [A+T])

GATCTACCACTGATA
ATACGTATCTAGTTA
GCTCGGGGCATGCC






PCR Primer Design
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.
Applications of PCR
Basic Research
Applied Research
Genetic matching
Detection of pathogens
Pre-natal diagnosis
DNA fingerprinting
Gene therapy


Mutation screening
Drug discovery
Classification of organisms
Genotyping
Molecular Archaeology
Molecular Epidemiology
Molecular Ecology
Bioinformatics
Genomic cloning
Site-directed mutagenesis
Gene expression studies
Applications of PCR
Molecular Identification
Sequencing
Genetic Engineering
Molecular Archaeology
Molecular Epidemiology
Molecular Ecology
DNA fingerprinting
Classification of organisms
Genotyping
Pre-natal diagnosis
Mutation screening
Drug discovery
Genetic matching
Detection of pathogens
Bioinformatics
Genomic cloning
Human Genome Project
Site-directed mutagenesis
Gene expression studies
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 (REVERSE TRANSCRIPTION)
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.
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 RT-PCR.
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 Pathogens

Molecular 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 (STRs)
the repeat region is variable between samples while the flanking regions where PCR primers bind are constant
7 repeats
8 repeats
www.cstl.nist.gov/biotech/strbase/ppt/4
Genotyping by STR markers
Molecular Identification:
A Homologous Chromosome Pair
One from mom
One from dad
What is the difference?
Image: [email protected]
13 CODIS Core STR Loci
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539
D18S51
D8S1179
D3S1358
FGA
VWA
AMEL
AMEL
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
Quantitative PCR can be used to determine the copy number 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).
Quantitative RT-PCR is often used to quantitate the level of 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.
- Khi 2 chất cho và nhận tách nhau thì tín hiệu nền là tính hiệu huỳnh quang của chất cho

- 2 probe bắt cặp với DNA đích: 1 probe mang ở đầu 3` chất cho fluorescein (fluorescein donor); 1 probe mang ở đầu 5` chất nhận fluorescein (fluorescein acceptor). Probe thứ 2 này chặn ở đầu 3` không cho Taq polymerase kéo dài mạch từ đầu này

- Ánh sáng chuyền từ chất cho đến chất nhận ? phát ra ánh sáng huỳnh quang màu đỏ
TaqMan™ assay
ỨNG DỤNG CỦA REAL TIME PCR:

Định lượng mức độ biểu hiện của gen
Đo sự hiện diện của DNA hay RNA trong các mẫu bệnh hay công nghiệp
Chẩn đoán lâm sàng tác nhân gây bệnh virus hay vi khuẩn (<1 h)

Summary
blood, chorionic villus, amniotic fluid, semen, hair root, saliva
68,719,476,736 copies
Gel Analysis, Restriction Digestion, Sequencing
1. simple
2. powerful
A. sensitive – sensitivity
B. specific – specificity
C. reliable – reliability; fidelity
3. fast
Three Aspects of PCR
Take home message
Thông tin liên quan đến PCR: các quá trình, thành phần và đặc điểm/ý nghĩa, tối ưu hóa phản ứng…
Ứng dụng của PCR
I-5-
70
Tài liệu tham khảo:
1/Basic Principles and Components of PCR. NSYSU
CHUNG-LUNG CHO
2/ PCR. Dr. Jason Linville
University of Alabama at Birmingham
[email protected]
3/ PCR of Chloroplast DNA. Kath Crawford
Jan Barfoot

The use of amplified chloroplast DNA (cpDNA) to investigate evolutionary relationships of common plants
Plant PCR
(i) Extraction of DNA from plant material (p8)
FTA card - chemically treated paper matrix for the safe collection, transport, storage, purification and analysis of DNA.
DNA is extracted on to the paper matrix, purified and then amplified.
Plant PCR
Place backing board between the back cover of the card and the absorbent layer.
Plant PCR
Place a piece of fresh plant material on to one box on the FTA card.
Ensure it does not extend outwith the box.
Close cover.
Plant PCR
Using a pestle, squash the leaf on to the card until moisture has soaked through to the back of the absorbent layer. Discard squashed plant material.
Plant PCR
Plant PCR
Repeat extraction for second plant material in a different box.
Try to ensure no `escape` to another box!
Plant PCR
Make sure that moisture from the leaf has soaked through to the back of the paper.
Plant PCR
Repeat extraction for third and fourth plant materials in the remaining boxes.
Try to ensure no `escape` of extracted materials to other boxes!
Plant PCR
Have you labelled your samples?
Plant PCR
Leave cards open to dry.
Transfer to lecture theatre.
One completed card (four samples), one punch per 8 people
Plant PCR
Use the punch in turn (cleaning between samples) so that each person removes one disc - the DNA to be amplified is on the disc.
One Brassica and one non-Brassica per pair.
One of each sample or one of three samples + negative control (blank paper, one per card) per four people.
(ii) Purification of the extracted DNA (p9)
Plant PCR
1. Place cutting/backing board (clean!) behind absorbent layer.
Place tip of the punch over the area to be sampled, press firmly and rotate to remove a paper disc. Important - choose an area where the extract has soaked through to the back.
Clean punch between samples by removing a disc of paper from an extract-free area.
Plant PCR
2. Use a cocktail stick to transfer the disc from the punch into a labelled, clear 1.5 cm3 microcentrifuge tube.
Use a different cocktail stick for each sample!
Twist dial to desired volume
Pick up pipette tip
Press plunger to first, soft stop
Insert pipette tip into solution to be transferred
Slowly release plunger to retrieve liquid
Move pipette tip to above desired well
Press plunger past first stop to second, hard stop to transfer liquid
Plant PCR
Plant PCR
3. Use a P-200 micropipette to add 150 ?l Purification reagent to the disc. Different tips for different discs!
4. Close tube and flick tube to wash the disc. Ensure the disc remains in the liquid.
5. Remove and discard purification reagent.
6. Repeat steps 3,4 and 5
Plant PCR
7. Use a fresh tip to add 150 ?l TE-1 buffer to the disc. Different tips for different discs!
8. Close tube and flick tube to wash the disc. Ensure the disc remains in the liquid.
9. Remove and discard buffer.
10. Repeat steps 7, 8 and 9
(iii) Amplification of cpDNA (p10)
1. Label PCR tube
2 - 4. Add reagents
4 ?l sterile deionised water
10 ?l CHc primer (yellow)
10 ?l CHd primer (blue)
PCR beads contain Taq polymerase, dNTPs, buffers, co-factors
Plant PCR
5, 6 Flick bottom of PCR tube - centrifuge if necessary.
Plant PCR
7. Use a clean cocktail stick to transfer the disc from microtube to PCR tube. Ensure disc is submerged in the PCR reagents.
8. Place in thermal cycler.
Plant PCR
Carry out pGLO transformation practical and then have lunch while DNA is amplifying.
(iv) Gel electrophoresis of PCR products (p 10) - One gel tank per pair
Plant PCR
1. Use a P-20 micropipette to add 2 ?l loading dye to 8 ?l DNA ladder (lilac microtube). Mix and load all 10 ?l into well 1 in a 1.5% agarose gel.
4. Repeat step 3 for other PCR samples. Each gel tank should contain a ladder, two Brassicas, two non-Brassicas and one other or a negative control.
Plant PCR
3. Load 10 ?l of the sample into a different well in the gel. Note sample/well!
2. Using a fresh tip, add 2 ?l loading dye to your amplified sample and mix.
Plant PCR
Plant PCR
Mechanical breakdown of cell walls/membranes by homogenisation with sand
Chemical disruption of cellular membranes by addition of detergent (SDS)
EDTA chelates Mg++ ions, helps break up protein complexes
NaCl helps disrupt cells and precipitate DNA
Tris buffer maintains appropriate pH
Ethanol to precipitate DNA
Plant PCR -extraction
Traditional method
Flinders Technology Australia, Fast Track Analysis
Commercially obtained from Whatmans -contain SDS, TrisEDTA buffer and other proprietary reagents
Application in research, diagnostics, environmental science, forensics and DNA databases
Included in SAPS/NCBE PCR kit
Plant PCR -extraction
FTA Cards - technology for processing nucleic acids invented by Professor Leigh Burgoyne of Flinders University
Commercial preparation, composition unknown but possibly contains SDS, Tris EDTA buffer
Removes naturally occurring agents that would inhibit the PCR. For example, compounds that contain heavy metals such as chlorophyll, and other proteins.
Purification buffer
Plant PCR -extraction
Removes SDS
Ensures correct pH
TE-1 buffer (10 mM TrisHCl,0.1 mM Na2EDTA pH 8)
Chloroplast DNA
Self-replicating DNA, 120 - 220 kb pairs
Highly conserved gene order
Contains genes that encode for tRNA (highly conserved across species)
? nucleotide sequences identical in the chloroplast DNA of almost all higher plants
?`consensus` or `universal` oligonucleotide primers
Plant PCR
Chloroplast DNA
higher frequency of mutations in non-coding stretches of cpDNA which lie between genes
? relatively high rates of evolutionary change
amplification of non-coding regions of DNA between genes may be used to show differences in the cpDNA of different populations
Plant PCR
PCR cycle
94�C for two minutes to ensure maximum separation of the strands
Thirty cycles of:
94�C for 30 seconds
55�C for 30 seconds
72�C for 45 seconds (at final stage 2 minutes)
PCR product can now be refrigerated or frozen
Plant PCR
ABI Prism 310 Genetic Analyzer
www.cstl.nist.gov/biotech/strbase/ppt/4
Close-up of ABI Prism 310 Sample Loading Area
www.cstl.nist.gov/biotech/strbase/ppt/4
ABI 310 Genetic Analyzer:
Capillary Electrophoresis
Amplified STR DNA injected onto column
Electric current applied
DNA separated out by size:
Large STRs travel slower
DNA pulled towards the positive electrode
Color of STR detected and recorded as it passes the detector
www.bioforensics.com
Forensic DNA Analysis
Forensic DNA Analysis
Black and white image of STR gel.
Samples will have one or two bands at each loci.
Gel Electrophoresis
Three generations of DNA testing
DQ-alpha
TEST STRIP
Allele = BLUE DOT
RFLP
AUTORAD
Allele = BAND
Automated STR
ELECTROPHEROGRAM
Allele = PEAK
From: www.bioforensics.com
Miniaturization of PCR
ST Lab-On-Chip
CÁC CẤU PHẦN CỦA PHẢN ỨNG
- Khuôn mẫu (Template): DNA cần khuyếch đại.
Ưu thế :
- Không cần sự tinh sạch cao, mẫu không được bảo quản tốt, bị phân hủy từng phần (vệt máu khô lâu ngày, tinh dịch, móng tay người chết.)
- Lượng mẫu giảm: từ 1�g - 100 ng
Mồi (amplifier; oligo hay primer) hợp lí cần:
- kích thước hợp lí, khoảng 18 và 25 nucleotide
- không có cấu trúc kẹp tóc, không có sự bắt cặp giữa mồi xuôi & mồi ngược
- Tm mồi xuôi & ngược không cách biệt quá xa
- thành phần nucleotide của các mồi cân bằng, tránh các cặp GC lặp lại nhiều lần
- đặc trưng cho trình tự DNA cần khuếch đại, không trùng với các trình tự lặp lại trên gen
- trình tự nằm giữa hai mồi xuôi & ngược không quá lớn, phản ứng PCR tối ưu trên những trình tự nhỏ hơn 1kb
ENZYME
Klenow của DNA polymerase I
Các DNA polymerase chịu nhiệt

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