Genetic Fingerprinting

Transcription

1 Genetic Fingerprinting

2 Introduction DA fingerprinting In the R & D sector: -involved mostly in helping to identify inherited disorders. In forensics: -identification of possible suspects involved in offences. -determining the maternity or paternity of an individual.

3 DA profiling developed by A. J. Jefferys in 1985 Humans have most of their DA in common Hair, nails, saliva, blood, semen etc. DA fingerprinting utilizes small differences called mini-satellites base pairs throughout the human genome which differ contain minute differences which make each person unique

4 H 2 P - CH 2 A deoxyadenosine 5 phosphate H - P - CH 2 H H 2 G deoxyguanosine 5 phosphate H H 2 T deoxythymidine 5 phosphate - P - CH 2 H H 3 C H C deoxyctyosine 5 phosphate - P - CH 2 H

5 H 2 P - CH 2 H A P - CH 2 H 2 G P - CH 2 H 3 C H T - P - CH 2 C H 3-5 phosphodiester linkages

6 5 end H 2 P - CH 2 H A P - CH 2 H 2 G P - CH 2 H 3 C H T - P - CH 2 C H 3-5 phosphodiester linkages 3 end

7 DA Fingerprinting Techniques used to distinguish between DA of individuals: Restricted fragment length polymorphism (RFLP) Short tandem repeats (STR) (DA sequencing, polymerase chain reaction) Two common techniques for separation and identification: Gel Electrophoresis Capillary Electrophoresis

8 DA Samples Most common DA sample are from blood or saliva, but any fluid or tissue containing DA is suitable. A reference sample can be extracted using a bucal swab.

9 Restricted fragment length polymorphism (RFLP) still used but slowly replaced by more sensitive and accurate methods. involves fragmenting DA with restriction enzymes. Restriction enzymes may be harvested from bacteria, and used specifically for DA dissection.

10 Restriction Endonucleases Example: TaqI (Thermus aquaticus) Derived from hot springs bacteria Allows for cleavage of double stranded DA at the phosphodiester bond Restriction enzymes cleave sequences which contain certain base-pairs Resulting in sticky and blunt end fragments Depiction of a sticky end splice Depiction of a blunt end splice

11 Restriction fragments lengths are distinct and measurable A small fraction varies from person to person Variable fragments are termed Restriction Fragment Length Polymorphs (RFLP s)

12 Examples of commonly used restriction enzymes 10

13 Gel electrophoresis separates the fragments on agarose gel (long DA) or polyacrylamide gel (short DA). DA fragments can count from 300 to base pairs. on gel: negatively charged DA fragments migrate to the positive end. DA ladder can be compared to others.

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15 Detection: Southern Blot gel soaked in a alkaline solution to denature DA. denatured DA blotted onto a nitrocellulose/nylon membrane. Incubation of the membrane (nitrocellulose) or exposition to UV light (nylon) for hybridization with a fluorescent or radioactive probe. DA detected by absorbance measurements or on an X-ray film (radioactivity/fluorescence).

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17 Probing (Hybridization) Uses labeled single stranded DA This anneals to DA which was separated Usually 32 P or bioluminescence probes

18 Why RLFP is becoming obsolete RFLP is a qualitative approach. large amounts of DA are required DA tends to degrade due to harsch experimental conditions. Gel electrophoresis long to run.

19 Short tandem repeats (STR) STR: repeated sequences of 3-5 base pairs (loci) which can be identified in a known database. useful in DA analysis because they show great variability among individuals. method yielding error rate of about 1 in does not require very much DA, can be coupled with PCR.

20 STR technology : evaluates specific polymorphic regions (loci) that are found on DA. In the US, the FBI has chosen 13 specific STR loci as standard. All forensic laboratories can then establish uniform DA databases and share forensic information. the likelihood that any two individuals (except identical twins) have the same 13-loci DA profile can be as high as 1 in 1 billion or greater.

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22 STR Analysis extraction of nuclear DA from the cells PCR amplification of the specific polymorphic regions analysis of these DA regions by real time PCR or CE Determination of the number of repeats of the STR sequence in question CE determination use fluorescent dyes

23 STR approach involving RFLP

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25 DA sequencing: the Sanger method Also called dideoxy, or termination method (inventor Frederick Sanger, 1980 obel prize in Chemistry). dideoxy : the technique uses synthetic nucleotides lacking H at the 3 carbon atom. A dideoxynucleotide, when added to the growing DA strand, stops elongation because there is no 3 -H for the next nucleotide to be attached to.

26 P - CH 2 P - H 2 CH 2 P - H CH 2 - H 2 P - H 3 C CH 2 H P - H CH 2 P - H 2 CH 2 P - H CH 2 H 2 CH 2 3 end is hydroxylated = continuation 3 end is deoxy = stop - P - H 3 C H A G T C

27 Procedure The DA to be sequenced is prepared as a single strand. This template DA is supplied with: a mixture of all four normal (deoxy) nucleotides triphosphates in ample quantities datp dgtp dctp dttp a mixture of all four dideoxynucleotide triphosphates, each present in limited quantities and each labeled with a "tag" that fluoresces a different : ddatp ddgtp ddctp ddttp DA polymerase I

28 Because all 4 normal nucleotides are present, chain elongation proceeds normally until, by chance, DA polymerase inserts a dideoxy nucleotide instead of the normal deoxynucleotide. If the ratio of normal to dideoxy nucleotides is high enough, some DA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.

29 At the end of incubation, the chains are separated according to length. A difference of one nucleotide is enough to separate strands from each other. Each dideoxynucleotide fluoresces at a different λ when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.

30 DA sequence of 455 nucleotides of the lysu gene of E. coli

31 Sequencing DA using the ion torrent TM method During the polymerisation of DA, release of H + (3 H) triggers an ion sensor, which generates a current. "sequencing by synthesis : a complementary strand is built based on the sequence of a template strand.

32 A microwell containing a template DA strand to be sequenced is flooded with a single species of deoxyribonucleotide triphosphate (dtp). If the introduced dtp is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers an ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence, multiple dtp molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.

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34 Michael Glen Becker, U of M, 2013

35 Polymerase Chain Reaction Developer: Kary B. Mullis obel Prize, 1993 Goal: To clone target DA sequences to have more for analysis Excellent for small amounts of DA PCR can amplify small, degraded DA samples, or restriction fragments Uses Taq Polymerase This enzyme is stable at high temperatures, as needed to denature DA

36 Three main steps: Denaturation of DA at about C Hybridization-annealing Taq polymerase reproduces the single strands into double strands The process is repeated several times to obtain a sufficient amount of DA for analysis.

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38 Segment of interest to be copied and tested Primer template Primer template Large DA chain from sample

39 5 3 A C B D 3 5 template complement 95 o C

40 5 A B 3 template 3 C D 5 complement denaturation 95 o C 5 A B 3 template 3 C D 5 complement

41 5 A B 3 template 5 A 60 o C Anneal 5 Primer C D complement 3

42 5 A B 3 template PCR 5 A B 3 C D complement 3

43 5 A B 3 denaturation 95 o C 3 C D complement

44 5 A D 60 o C 3 Anneal B 3 Primer 3 C D complement

45 C D A B 5 3 PCR

46 C A B C D