DNA fingerprinting 7 jan 2015

DNA fingerprinting 7 jan 2015

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  1/9/2015 DNA fingerprinting1 DNA FINGERPRITING DNA PROFILING DNA TYPING By Dr Ichha Purak University Professor Department of Botany Ranchi Women’s College,Ranchi
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  Professor Sir AlecJeffreys, Kt, FRS: Discovered DNA fingerprinting in 1984 Professor of Genetics and Royal Society Wolfson Research Professor, Department of Genetics, University of Leicester. 1/9/2015 DNA fingerprinting 2
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  HISTORY Professor Alec Jeffreysin 1984 developed the DNA(Genetic) fingerprinting process at Leicester University which is one of his best contributions to human genetics Since discovery DNA fingerprints are used for identifying individuals, establishing family relationships, in medical research, forensic science for identification of criminals, paternity and immigration evidence. The purpose of DNA fingerprinting can also be extended to breeding plants and animals, conserving nature and understanding evolutionary processes. In 1987 it was used for the first time in law court of England to identify a victim in Rape case .DNA fingerprinting was first used as forensic evidence in 1988 1/9/2015 3DNA fingerprinting
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  ORDINARY FINGER PRINTSPRESENT AT FINGER TIPS 1/9/2015 DNA fingerprinting 4 Every person can be identified by his fingerprints and therefore thumb impression is used in place of signature for those persons who are unable to read and write. As fingerprints are unique so are being used for identification in forensic science since long ( 1930’s or even earlier)
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  As DNA ofevery individual consists of millions of bases and every person has a different sequence. It is not practicable to analyse the complete genome sequence so for DNA finger printing some repeated sequences can be analysed which can be isolated and separated by Gel electrophoresis The sequence of DNA fragments such as Minisatellite or VNTRs, Microsatellite or STR or RFLP can be obtained by southern blotting technique so can be employed for DNA finger printing. RFLPs (Restriction Fragment Length Polymorphism ) are DNA fragments produced when DNA is digested by Restriction endonucleases which are able to cut phospho di-ester bond of DNA at specific sequences 1/9/2015 DNA fingerprinting 5
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  Variable number tandemrepeats, or VNTRs represent specific locations on a chromosome in which tandem repeats of 9-80 or more bases repeat a different number of times between individuals and also variation in length. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. VNTRS are readily analyzed using the RFLP approach and a probe specific to a VNTR locus. Their analysis is useful in Genetics and DNA fingerprinting for comparative analysis in forensics. Currently the most popular method of DNA fingerprinting is done by using Short term Repeats (STRs or Microsatelite ) which have repeat sequences of only 2-5 base pairs . Since the length of DNA fragment being analyzed is short enough can be amplified by polymerase chain reaction (PCR) 1/9/2015 DNA fingerprinting 6
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  Variable Number ofTandem Repeats (VNTR) AGTTCGCGTGA AGTTCGCGTGA AGTTCGCGTGA AGTTCGCGTGA AGTTCGCGTGA Repeat sequence length: 10-100 base pairs/repeat _____________________________________________________ ______ Short Tandem Repeats (STR) ATGCC ATGCC ATGCC ATGCC ATGCC Repeat sequence length 2-9 base pairs/repeat VNTR AND STR 1/9/2015 7DNA fingerprinting
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  PRINCIPLE OF DNAFINGER PRINTING VNTR is a tandem repeat from a single genetic locus in which the number of repeated DNA segments varies from individual to individual but are inherited and are used for identification purposes as in DNA fingerprinting. The VNTRs of two persons may be of same length and sequence at certain sites, but vary at others. A child might inherit a chromosome with tandem repeats both from mother and father, so VNTR alleles of a child resemble to both parents. Southern Blot is performed to determine if a person has a particular VNTR. Southern Blot is probed through a hybridization reaction, with a radioactive version of the VNTR being analysed. The pattern which results from this process is often referred as a DNA fingerprint 1/9/2015 DNA fingerprinting 8
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  STEPS INVOLVED INDNA FINGER PRINTING ISOLATION RESTRICTION ENDONUCLEASE DIGESTION GEL ELECTROPHORESIS  DENATURATION SOUTHERN BLOTTING USING VNTR/STR/ RFLP MAKING A RADIOACTIVE PROBE (SSDNA ) CREATING HYBRIDIZATION REACTION DETECTION OF HYBRIDIZATION BY AUTORADIOGRAPHY OR OTHER METHODS 1/9/2015 DNA fingerprinting 9
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  STEPS OF DNAFINGER PRINTING 1/9/2015 DNA fingerprinting 10
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  AB C D 1/9/2015 DNAfingerprinting 11
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  SAMPLE REQUIREMENT FORDNA FINGERPRINTING For DNA fingerprinting only a small amount of tissue like blood, semen, skin, vaginal swab or even follicle root of hair or plant tissue is needed as only a small amount of DNA is sufficient. DNA can be isolated even from bone marrow of old bones of victims or blood stains from clothings or discharge can serve the purpose. Typically DNA content of about 10,000 cells or I microgram is sufficient. If amount is less(less than 1 µg) it can be amplified by Polymerase Chain Reaction (PCR) DNA AMPLIFICATION BY POLYMERASE CHAIN REACTION PCR is an invitro procedure developed by Kary Mullis (1985) that amplifies enzymatically a particular DNA sequence flanked by two oligonucleotide DNA primer. 1/9/2015 DNA fingerprinting 12
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  For this aapparatus Thermal cycler is used which has provisions to alternate temperature in cyclic manner. PCR is based on semi conservative DNA Replication PCR involves following steps Denaturation step takes place at 94-98ᵒC. Double stranded DNA is denatured by dissolving hydrogen bonds between base pairs resulting in two single stranded structure which act as template Annealing step takes place at 50-65ᵒ C for annealing of DNA primers Extension or Elongation step DNA synthesis takes place at 72ᵒ C by using Taq DNA polymerase and all the 4 dNTPs resulting in formation of two DNA double Helices. These three steps are cycled repeatedly (20-35-40) resulting in making copies of DNA exponentially as per requirement 1/9/2015 DNA fingerprinting 13
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  STEPS INVOLVED INDNA FINGER PRINTING 1/9/2015 DNA fingerprinting 16  DNA is extracted from cells in high speed refrigerated centrifuge using detergents to remove debries.  DNA is cut into fragments by using one or more site specific Restriction Endonucleases and RFLPS( Restriction Fragment Length Polymorphism or Fragments of different size ) are obtained .  Choped DNA fragments are passed through Agarose Gel Electrophoresis or Polyacrylamide Gel Electrophoresis (PAGE)  The separated fragments can be visualized by staining them with Ethidium Bromide dye that fluorescence under U-V radiation. Bromophenol Blue is used as tracking dye.  Double stranded DNA is then split into single stranded DNA using alkaline chemicals
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  These separated singlestranded DNA fragments are then transferred to Nitrocellulose or Nylon sheet placed over the gel.  The adherence ( Blotting ) of single stranded DNA fragments to nitrocellulose sheet is called Southern Blotting This protocol of adsorbing DNA was invented by the Scientist E M Southern.( 1976)  Nitrocellulose sheets are baked at high temperature so as to fix single stranded DNA to it. The nitrocellulose sheet is then immersed in a bath and radioactive probe (DNA ) a synthetic oligonucleotide DNA segment with known sequence are added  The probe targets a specific nucleotide sequence which is complementary (VNTRs Sequence ) and hybridize them  Finally NC sheet ,containing the SSDNA and Radioactive probe is exposed to autoradiography . Dark bands develop at the site of hybridization and help in identifying Hybridization or homology 1/9/2015 DNA fingerprinting 17
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  DEVELOPMENT OF RADIOACTIVEPROBES BY JEFFEREYS In between the genes which code for protein synthesis are intervening non coding regions.These are called hypervariable regions because they vary from person to person.No two people, except identical twins, share same set of regions. In the hypervariable regions a simple sequence of 10-15 bases called a core sequence can be repeated over and over again. In 1984 Jeffreys discovered that core sequence could be used as genetic markers for the hypervariable regions. Jeffreys isolated two core sequences of DNA and copied them many times in the laboratory to produce large quantities of markers which he labeled with radioactive chemicals. Now these genetic markers could be used as genetic probes The probes can be attached to core sequences in a sample of DNA to find the position of hypervariable regions using photographic film to detect the radioactivity. 1/9/2015 DNA fingerprinting 18
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  1/9/2015 DNA fingerprinting19 The probes used for DNA fingerprinting are usually prepared from mini – satellite DNA which is highly variable mainly due to variation in number of tandem repeats of short core sequence. These probes hybridize under condition of low stringency , to a number of polymorphic loci represented by the mini-satellite DNA. The regions of mini satellite may occur dispersed throughout the human genome or may be clustered onto a single chromosome. A simple universal probe, a tandem repeat of GATA, has been developed from sex chromosome of branded Krait. This probe is reportedly very useful in DNA fingerprinting of man and many other organisms
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  MAKING A RADIOACTIVEPROBE For making a radioactive probe ,the double stranded DNA to be used to make probe is put in a tube . A nick is induced in one of the strand and to this DNA polymerase enzyme and all the four deoxyribonucleotides are added of which CMP is labeled by having 32P. DNA polymerase strarts repair at the point of nick and old bases are replaced by new base. Radioactive C is added where there is G on other strand (Template ) . After repair the Double stranded DNA is subjected to denaturtion. After denaturtion one strand has label and other is Non-labeled 1/9/2015 DNA fingerprinting 20
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  1 2 3 4 5 MAKINGA RADIOACTIVE PROBE Nick 1/9/2015 DNA fingerprinting 21
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  CREATING A HYBRIDIZATIONREACTION Hybridization is the coming together, or binding, of two genetic sequences. The binding occurs because of the hydrogen bonds between base pairs. ( A & T and C & G ) DNA must first be denatured, usually by using heat or chemicals. Denaturing is a process by which the hydrogen bonds of the original double-stranded DNA are broken, leaving a single strand of DNA whose bases are available for hydrogen bonding. 1/9/2015 DNA fingerprinting 22
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  A single-stranded radioactiveprobe can be used to see if the denatured DNA contains a sequence homologous to that of probe . For this the membrane with denatured DNA strands is put into plastic bag along with probe and some saline liquid or buffer and is allowed to make base pairs between complementary sequences 1/9/2015 DNA fingerprinting 23
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  Partial or completehybridization depends on varying homologous sequences to the probe sequence .Hydrogen bonds will be established between A & T by 2 H bonds and G & C by 3 H bonds where the sequence is homologous 1/9/2015 DNA fingerprinting 24
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  The DNA FINGERPRINTINGPROCESS 1/9/2015 DNA fingerprinting 25
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  Applications of DNAFingerprinting DNA fingerprints are useful in several applications of human health care research, as well as in the justice system DNA Finger Printing can be effectively used in forensics to identify or match the DNA of suspected individuals in crimes such as murder, rape and in paternity and immigration disputes 1/9/2015 DNA fingerprinting 26
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  PATERNITY AND MATERNITY DNAFingerprinting can be used to establish paternity and maternity on the basis of VNTR pattern because a person inherits his or her VNTRs from parents. For this VNTR pattern of Mother ,Father and child or children are compared . By this disputes of Biological father can be solved Parent-child VNTR pattern analysis has been used to solve standard father identification cases as well as more complicated cases of confirming legal nationality and in instances of adoption of biological parenthood As VNTR patterns are inherited ,can be used to establish family relations. Many people choose to trace their heritage by using and tracking DNA fingerprints. DNA fingerprinting was also used in an effort to identify a missing Russian princess Anastasia Romanov through remains found in a family grave. 1/9/2015 DNA fingerprinting 27
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  Here is anexample of a VNTR. In the diagram, D1 is a biological daughter, D2 is a step-daughter(daughter of the mother and an x- husband),S1 is a biological son and S2 is an adopted son. 1/9/2015 DNA fingerprinting 28
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  Criminal identification andForensic Evidence DNA isolated from blood, hair root follicles, skin cells, spots on clothing or other genetic evidences left at the crime scene (murder or rape) can be compared through VNTR patterns, with the DNA of a criminal suspect (S) to determine or identify the criminal victim and give relief to non-victim DNA fingerprinting was first used as forensic evidence in the murder conviction of Colin Pitchfork in 1988. Since then, crime scene identification, criminal identification and victim identification is widely done by matching DNA obtained from hair root follicle, blood, semen, skin and other human body parts and secretions. Many prisoners have been released based on new evidence provided through DNA fingerprinting. 1/9/2015 DNA fingerprinting 29
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  DNA fingerprints of7 suspected victims compared with that of blood stain obtained from murder scene. On the basis of DNA bands Number 3 appears as victims 1/9/2015 30DNA fingerprinting
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  Personal Identification: DNAcan be used to identify individual persons , but this doesn’t seem practical as the process is too complex and having a DNA database is expensive DNA Finger printing can be used for personal identification as social security number, photo identity , Blood group, notable phenotypic traits etc. The technology of profiling individuals based on VNTR patterns is very expensive. Because every organ or tissue of an individual contains the same DNA fingerprint, the U.S. armed services have just begun a program to collect DNA fingerprints from all personnel for use later, in case they are needed to identify casualties or persons missing in action. The DNA method will be far superior to the ordinary finger prints, dental records, and blood typing strategies currently in use. 1/9/2015 DNA fingerprinting 31
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  Immigration Evidence According tothe Dolan DNA Learning Center, a mother and son were separated by immigration officials, The dispute was solved by court in 1985 when DNA fingerprint evidence proved that the two were mother and son and belong to their own country of England. Unidentified immigrants can be assigned to their official country . Medical Research DNA fingerprinting helps in the early detection of inherited diseases and their treatment. Through the study of genetic patterns of individuals and groups, medical research can potentially lead to cures for inherited diseases. Another medical research purpose for DNA fingerprinting is to match recipients of live organs from the donors, making transplants more successful. 1/9/2015 DNA fingerprinting 32
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  Developing Cures forInherited Disorders Research programs to locate inherited disorders on the chromosomes depend on the information contained in DNA fingerprints. By studying the DNA fingerprints of relatives who have a history of some particular disorder, or by comparing large groups of people with and without the disorder, it is possible to identify DNA patterns associated with the disease in question. This work is a necessary first step in designing an eventual genetic cure for these disorders. 1/9/2015 DNA fingerprinting 33
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  Diagnosis of InheritedDisorders DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn babies in hospitals around the world. These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others. Early detection of such disorders enables the medical staff to prepare themselves and the parents for proper treatment of the child. In some programs, genetic counselors use DNA fingerprint information to help prospective parents understand the risk of having an affected child. In other programs, prospective parents use DNA fingerprint information in their decisions concerning affected pregnancies 1/9/2015 DNA fingerprinting 34
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  Miscellaneous Purposes Through DNAfingerprinting, selective breeding of animals and plants in possible. According to a U.S. Department of Agriculture and Cooperative Extension Service publication, "a scientist who knows which piece of DNA is associated with a desirable trait can select plants or animals that have that piece of DNA. It is often easier and less expensive to select plants or animals that have the DNA marker than to grow them to maturity and see if they develop the desirable trait.” 1/9/2015 DNA fingerprinting 35 DNA Fingerprinting can be used to identify racial groups to rewrite biological evolution It was also used for identification of the body of Nazi physician Joseph Mengele, the so-called "Angel of Death.” By DNA fingerprinting a kid lost in Tsunami was handed over to his actual parents by comparing DNA of other claimants.
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  Recent Developments andthe Future prospects of DNA Testing) (Profiling ) Databases Many US states have planned to make database of DNA profiles of individuals having criminal record New profiling methods Every cell of human being contain 23 pairs of chromosomes so each gene has two copies. In addition that each cell has organelles as mitochondria . Mitochondrial DNA can be used for DNA fingerprinting when amount of biological samples are limited in case of disaster or accident or burning ,the bodies becomes badly destroyed. 1/9/2015 DNA fingerprinting 36 END OF PRESENTATION