DNA: A New Forensic Science Tool

Principles of Gene Manipulation ·
Created almost exclusively by
Jennifer Cooper · America Madrigal · Laleña Vellanoweth
PCR:
The Polymerase Chain Reaction (PCR) and Its Applications
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PCR: PCR and Its Applications
Definition of PCR

Requirements for PCR

PCR Process
A. Denaturing Stage
B. Annealing Stage
C. Extending Stage
D. Replication

Some Applications of PCR
A. Library Screening
B. Forensic Identification
C. Making a probe
D. Subcloning

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PCR What is it?
The Polymerase Chain Reaction (PCR) is an
in vitro method to amplify a specific region of DNA.

PCR is extremely sensitive, with the capability
of amplifying minuscule quantities of DNA.
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PCR Why do it?
study DNA from fossils
compare forensic samples
identify remains
clone subfragments that don’t have convenient restriction sites
unite living members of a separated family
determine tissue type for transplants
amplify ds cDNA fragments from the reverse transcription products of mRNA (RT-PCR)
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PCR REQUIREMENTS
DNA sample
· very small amounts (ng or sometimes less) if DNA is in good shape
· may be able to use DNA from only one cell
· only a few molecules must be intact
samples with larger numbers of molecules can be in poor shape or degraded

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PCR REQUIREMENTS (cont’d.)
Two primers
· flank region you are interested in
· you must know the sequence of the flanking regions so you can order
appropriate primers
Heat stable polymerase
Four dNTPs
Thermocycler (standard, but optional)
· changes temperature very rapidly for each cycle (denature, anneal, extend)
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Programmable thermocycler
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Same thermocycler - open.
Notice heating block with three samples in pink tubes.
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PCR METHOD
There are four basic steps in PCR

1. Denaturing Stage
2. Annealing Stage
3. Extending Stage
4. Replication

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PCR METHOD – DENATURING STAGE
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(reference: www.library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)
PCR METHOD – ANNEALING STAGE
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(reference: www.library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)
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PCR METHOD – EXTENDING STAGE
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(reference: www.library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)
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PCR METHOD - REPLICATION
Each cycle doubles the copies of double stranded DNA.



DNA




Cycle 1 2 3 4 5 …

The average number of cycles performed with PCR, for efficiency reasons, is thirty.
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PCR APPLICATIONS - library screening
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The next three figures illustrate screening a library for your favorite gene.
You may have designed primers to try based on cDNA sequence which you have already determined.
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PCR APPLICATIONS
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2. Forensic Identification (fingerprinting):
· Takes advantage of known VNTR loci in human DNA.
· VNTR’s are Variable Number of Tandem Repeats
· STRs are a subset of VNTRs.
The repeats are very Short.
· The number of repeats at any one loci vary among the population.
- Close relatives have the same number of repeats
- Flanking sequences of STR’s at many loci are known.

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Any one individual will have only one or two PCR lengths (homo- or heterozygous).
Short differences can be resolved by capillary electrophoresis.
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Example of STR PCR products run on an agarose gel.
These products are sufficiently different in length to discriminate by gel electrophoresis.
13 CODIS Core STR Loci with Chromosomal Positions
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539
D18S51
D8S1179
D3S1358
FGA
VWA
AMEL
AMEL
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Fingerprinting points
The greater the number of different primer sets used in fingerprinting, the less likely the chance of a random match.
The greater the variation at each locus within a population, the more useful the locus is in making discriminations.
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For more information,
check out the slide lecture on STR-PCR
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PCR APPLICATIONS – making a probe

Problem
Imagine that you want to make a probe representing one end of a cDNA you have cloned. There are no convenient restriction sites, and you do not want to use an RNA probe. Describe how you could use PCR to make the probe.
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Answer
Pick the region you want to use.
Design primers complementary to the two ends of that region.
Be careful they go in the correct direction!

Set up PCR, incorporating a labeled nucleotide.
Isolate product and use as a probe.
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PCR APPLICATIONS - subcloning

Imagine that you want to clone the promoter region of the ovalbumin gene from -600 to the start site of transcription into the vector shown below.
You want the promoter to be inserted in the correct orientation so that it can direct transcription of the luciferase gene.
Choose a PCR-based strategy that will allow you to get the region you want into the vector in the orientation you want it to be.
Make your plan so that you do not need to use adaptors.
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Luciferase gene
A Reporter Vector
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PCR APPLICATIONS
Clone or Subclone:
· Take a region from within a larger one and clone it into a vector of choice.
· Design your primers to flank the region you want to subclone.
a) Include an appropriate restriction enzyme (RE) recognition sequence in each of your primers
your product will have RE sites at the ends, allowing you to cut and ligate it into a prepared vector.
or
b) Taq polymerase leaves 3’ single “a” overhangs, therefore your vector can be prepared with a single “t” overhang. This process is called T/A cloning. This will allow you to ligate your product into a prepared vector.
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Luciferase gene
A Reporter Vector
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Design primers with RE sites at ends
Sac I
Hind III
Complementary to one end of YFRegion
Complementary to the other end of YFRegion
When designing primers, you add additional nucleotides at each of the 5’ ends and incorporate the RE sites of your choice in the sequence.
Addn’l nucs specifying RE site
Addn’l nucs specifying RE site
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*
*
* Fragments of desired length with desired RE sites! More each round!
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PCR REFERENCES
l. Lecture notes from Dr. Sharp on 11/03/00 and 11/06/00.
2. Gene Cloning: an introduction. T.A. Brown
3. The world wide web:
http://sunsite.berkeley.edu/PCR/whatisPCR.html
http://www.accessexcellence.org/AB/GG/polymerase.html
http://www.sciam.com/1998/0598issue/0598working.html
http://www.faseb.org/opar/bloodsupply/pcr.html
http://faculty.plattsburgh.edu/donald.slish/PCR.html
http://library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html
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After a long and tedious process, this PowerPoint presentation has now ended. We hope it is of good help in your studies.