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 H2 P - CH 2 A deoxyadenosine 5 phosphate H - P - CH 2 H H2 G deoxyguanosine 5 phosphate H H2 T deoxythymidine 5 phosphate - P - CH2 H H3C H C deoxyctyosine 5 phosphate - P - CH 2 H

5 H2 P - CH 2 H A P - CH 2 H2 G P - CH2 H 3C H T - P - CH 2 C H 3-5 phosphodiester linkages

6 5 end H2 P - CH 2 H A P - CH 2 H2 G P - CH2 H 3C 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) Techniques for separation and identification: Gel Electrophoresis Capillary Electrophoresis Fluorescence + ext generation methods

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 (this step is still used in many approaches). 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 in length.

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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 if coupled to PCR.

20 STR : evaluates specific polymorphic regions (loci) that are found on DA. the FBI has identified 13 specific STR loci as standards. All forensic labs 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.

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22 STR Analysis extraction of nuclear DA from the cells targeted PCR with fluorescent primer analysis of amplified DA segment by RFLP determination of the number of repeats of the STR sequences

23 STR approach involving RFLP

24 Polymerase Chain Reaction Developer: Kary B. Mullis obel Prize, 1993 Goal: Clone target DA sequences to have more for analysis or other 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

25 Main steps: Denaturation of DA at about C (opening of double-stranded) START primers annealed (60 o C) Taq polymerase makes complementary strands from START primers The process is repeated several times to obtain a sufficient amount of DA.

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27 Double stranded segment to be copied and sequenced Primer section Primer section Large DA chain from sample

28 5 A B 3 template 3 C D 5 complement denaturation 95 o C 5 A B 3 template 3 C D 5 complement

29 5 A B 3 template D START 5 60 o C Anneal 5 Primer 5 A B 3 template C D START PCR 3 5

30 5 A B 3 template C D complement START 3 5 Denaturation 95 o C

31 C D complement Anneal 3 5 START 5 A C D PCR 3 5 START A B 5 3

32 C A B C D C A B C D C A B C A B C D C D C A B C D

33 Michael Glen Becker, U of M, 2013

34 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.

35 P - CH 2 P - H2 CH 2 P - H CH2 - H2 P - H 3C CH 2 H P - H CH 2 P - H2 CH 2 P - H CH2 H2 CH 2 3 end is hydroxylated = continuation 3 end is deoxy = stop - P - H 3C H A G T C

36 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

37 Chain elongation proceeds normally until DA polymerase inserts a dideoxynucleotide instead of the normal deoxynucleotide. If the ratio of normal to dideoxynucleotides is high enough, some DA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.

38 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.

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

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42 DA polymerase catalyzes the incorporation of fluorescently labeled deoxyribonucleotide triphosphates (dtps) into a DA template strand during sequential cycles of DA synthesis. During each cycle, at the point of incorporation, the nucleotides are identified by fluorophore excitation.

43 Four basic steps: 1. Library Preparation. The sequencing library is prepared by random fragmentation of the DA sample, followed by 5 and 3 adapter ligation. Adapter-ligated fragments are then PCR amplified and gel purified.

44 2. Cluster Generation The library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. Each fragment is then amplified into distinct, clonal clusters through bridge amplification.

45 3. Sequencing Illumina detects single bases as they are incorporated into DA template strands. Four reversible terminator bound dtps are present during each sequencing cycle, but attach only transiently. The result is highly accurate base-by-base sequencing.

46 4. Data Analysis The newly identified sequence reads are aligned to a reference genome.

47 Genetic fingerprinting involves different combinations of methods e.g.: FRLP + PCR + gel electrophoresis + fluorescence labeling FRLP + fluorescence-pcr + gel electrophoresis FRLP + PCR targeted for STR + Illumina (polymerase+sequencing) PCR of segment of interest from full DA + ion torrent Depending on: length and detail of DA under investigation cost availability of technology reporting deadline