DNA analysis. Anja Bye Post doktor. K.G. Jebsen Senter for Hjertetrening. Institutt for Sirkulasjon og Bildediagnostikk Det Medisinske Fakultet NTNU

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1 DNA analysis Anja Bye Post doktor K.G. Jebsen Senter for Hjertetrening Institutt for Sirkulasjon og Bildediagnostikk Det Medisinske Fakultet NTNU

2 Focus of this lecture What is DNA? Comparing DNA from different sources Size separation of DNA by gel electrophoresis Forensic DNA analysis Paternity testing DNA analysis in disease DNA analysis in research

3 What is DNA? DNA: Deoxyribonucleic acid DNA is the hereditary material in humans Most DNA is located in the cell nucleus (nuclear DNA), but a small amount of DNA can also be found in the mitochondria (mitochondrial DNA) The information in DNA is stored as a code made up of four chemical bases: adenine (A) guanine (G) thymine (T) cytosine (C)

4 What is DNA? DNA bases pair up with each other, A with T and C with G, to form units called base pairs Each base is also attached to a sugar molecule and a phosphate molecule Together, a base, sugar, and phosphate are called a nucleotide Nucleotides are arranged in two long strands that form a spiral called a double helix The structure of the double helix is somewhat like a ladder

5 What is DNA? Human DNA consists of about 3 billion bases >99 % of the bases are the same in all people <1% of the DNA is responsible for the differences from person to person (length, hair color, risk of diseases etc) A large percentage of the human genome is described as Junk-DNA because it appears to have no biological function The order, or sequence, of these bases determines the information available for building and maintaining an organism, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences The DNA is organized in 23 pairs of chromosomes Every person inherits one set of 23 chromosomes from the mother and one set of 23 chromosomes from the father

6 Analyzing DNA DNA analysis is useful in several different situations: Comparing DNA from different sources Forensics (to tie a person to a crime) Paternity testing (to test whether people are related) In the clinical setting: Analyze particular parts of the DNA for identification or predicting the risk of inherited disease In medical research: Comparing DNA from sick and healthy individuals to find potential targets for gene replacement therapy and gene testing

7 Comparing DNA from different sources One way to identify whether DNA from two different sources are similar, is by breaking up the DNA strand into smaller pieces by enzymes called restriction enzymes Restriction enzymes do not break up the DNA molecule randomly but cut it at particular sites The technique is termed restriction fragment length polymorphism (RFLP)

8 For example, a restriction enzyme called EcoR1* recognises the base sequence CAATTC and cuts it between the two As recognised --C-C-G-C-A-G-C-T-G-T-C-A-A-T-T-C-T-C-T-C-C-G-G-A-T-C-C-A --C-C-G-C-A-G-C-T-G-T-C-A cut A-T-T-C- T-C-T-C-C-G-G-A-T-C-C-A- The fragments will have different length from individual to individual based on the different amounts of junk- DNA between the cuts To be able to visualize the length of the DNA fragments we can use a method called gel electrophoresis

9 Gel electrophoresis A technique used to separate DNA fragments by size Different samples are added to a gel which is subjected to an electrical field DNA has a negative charge and moves slowly towards the positive end The shorter fragments travel through the gel faster than the longer fragments DNA is visualized using fluorescent dyes

10 well DNA extract added gel solution

11 Voltage supply Negative electrode Positive electrode + DNA fragments move from negative to positive

12 Appearance of separated fragments on gel These bands will contain the shorter DNA fragments These bands will contain the longer DNA fragments Prof. E. Wood The pattern of bands in the gel is known as a genetic fingerprint starting positions

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14 Forensic DNA Analysis If DNA found in a sample of blood or other tissue at the crime scene matches the DNA of a suspect, this can be used as evidence J A DNA sample can be obtained from the suspect using blood, cheek epithelial cells taken from the mouth or even from the cells clinging to the root of a hair A variety of substances contains DNA, including blood, semen, saliva, hair, urine, bone, teeth, feces, and soft tissues

15 Reliability of DNA-analysis Suppose that.there is a chance of 1 in 10 that this fragment occurs in many individuals and there is a chance of 1 in 20 that this fragment occurs in many individuals and there is a chance of 1 in 10 that this fragment occurs in many individuals and there is a chance of 1 in 30 that this fragment occurs in many individuals, but

16 the probability of all 4 bands matching in any person other than the suspect is 1 in 10 x 1 in 20 x 1 in 10 x 1 in 30 = 1 in 10 x 20 x 10 x 30 That is 1 in 60,000 When a larger number of bands is involved, the probability that the suspect is not guilty becomes one in many thousands

17 Polymerase chain reaction (PCR) Often DNA samples obtained from crime scenes are small in quantity or degraded by sunlight or high temperature These samples are subjected to PCR, which is a valuable technique because it provides a millions of copies of small regions of DNA

18 Polymerase chain reaction (PCR)

19 Mitochondrial DNA sequencing Analysis of the DNA located in the mitochondria Unlike nuclear DNA, which only exists inside the nucleus of a cell, mtdna is found in multiple copies outside the nucleus This technique is valuable because mitochondria can serve as a useful source of genetic material when nuclear DNA samples are unavailable or degraded

20 The Colin Pitchfork Case Two young women raped and murdered in Narborough, England 5000 local men were asked to provide blood/saliva samples The first convicted of murder based on DNA fingerprinting evidence (1987)

21 The Unabomber Ted Kaczynski was a child prodigy accepted to Harvard University at the age of 16 In 1971, he moved to a remote cabin without electricity or running water to become self-sufficient He decided to start a bombing campaign after watching the wilderness around his home being destroyed by development The bombing lasted nearly 20 years, killing three people and injuring 23 others After years of investigation finally Using saliva, the FBI were able to match DNA samples from letters mailed to relatives by Kaczinski with DNA obtained from stamps on letters mailed by the Unabomber

22 The Combined DNA Index System (CoDIS) A database of DNA profiles from violent felons and crime scene samples The database currently contains about felons and crime scene profiles (19,00 hits) Extremely powerful investigative tool, linking crimes, and pulling suspects out of thin air!

23 Paternity testing DNA analysis that establish genetic proof as to whether a man is the biological father of an individual The current techniques for paternal testing are using PCR and RFLP

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29 DNA Fingerprinting & Paternity Male 1 is the father. His DNA fingerprint is like that of the child except for the portion supplied by the mother. 29

30 DNA analysis in disease Today there are several commercially available DNA test for clinical use Huntington's disease Cystic fibrosis Sickle cell anemia DNA tests that determines inherited predisposition to certain types of breast cancer and Alzheimer's disease As more is learned about the information stored in DNA, DNA tests may be used more widely in preventative medicine to help individuals avoid specific foods or certain environmental conditions

31 Sickle cell anemia Characterized by red blood cells that assume an abnormal, rigid, sickle shape Occurs because of a mutation in the hemoglobin gene, causing one of the several amino acids to change Life expectancy is shortened, with studies reporting an average life expectancy of 42 in males and 48 in females

32 Sickle cell anemia

33 Breast cancer Breast cancer is the most common cancer among women In 1990, Mary-Claire King and associates localized the first major susceptibility gene called BRCA1 (Breast Cancer) BRCA2 was localized in 1995 Both are also linked to cases of ovarian cancer When BRCA1 has normal function it works by suppressing tumor growth Mutation on BRCA1 and/or BRCA2 are believed to account for 5-10 % of all cases of breast cancer

34 Standard BRCA-1 and BRCA-2 tests detect common mutations in the genes, mutations that are known to increase the risk of breast and ovarian cancer development

35 The BRCA1 Gene Normally Encodes a Protein that Regulates Cell Growth Cell Nucleus Chromosomes BRCA1 Gene BRCA1 Protein which keeps cell growth in check

36 A mutated BRCA1 gene produces a faulty protein which allows cells to grow too quickly Cell Nucleus Chromosomes BRCA1 Gene Mutation A An non-functional absent or non-functional BRCA1 Protein BRCA1 which protein cannot which keep cannot keep cell cell growth in in check

37 Cancer Risks for a woman who carries a mutation in BRCA1 Life time risk of breast cancer ~85% 10 year risk of a second breast cancer ~35%-70% Life time risk of ovarian cancer ~50% Probable increased risk of other cancers (eg, prostate, colon)

38 DNA analysis in research Comparing parts of the DNA from a large group of subjects with a certain disease with a large group of healthy subjects Aim: To find parts of the DNA that differ between sick and healthy subjects Overall aim: To find potential targets for gene replacement therapy and/or gene testing Common used method: Analyzing a large number of single nucleotide polymorphisms (SNPs)

39 Single Nucleotide Polymorphisms (SNPs) A difference in one single base at a particular place on the DNA strand which is found in <1% of the population 1.8 million identified in human genome Several of these are likely to be the cause of disease Microarray technology allow us to detect over 1 million SNPs in one single sample

40 Other applications for DNA analysis DNA analysis is now a common tool for identify remains after tragedies such as airline accidents and big fires Anthropologists are using DNA analysis to study the migration of human beings across the oceans Historians are employ these techniques to identify genetic disease in famous individuals

41 Consumer genetics Recently it has become quite popular to get your own DNA analyzed in an easy and cheap way You can simply contact one of the companies on-line and they will provide information and equipment for you to collect your own DNAsample (example: The company will then provide you with an risk estimation for common diseases, check if you are a carrier of genetic diseases, check you drug response, as well as provide information of other interesting traits

42 Example from 23andme

43 Example from our research group At the K.G. Jebsen Center for Exercise in Medicine we are currently analyzing DNAsamples collected in the HUNT-study Our goal is to find variations in DNA (SNPs) that determines whether you are born with high or low fitness level A persons fitness level is suggested to be about 50 % inherited (that is, determined by your DNA sequence) As low aerobic fitness level is closely correlated to cardiovascular mortality, we hope that DNA-variations associated with low aerobic fitness can represent targets for the prevention and treatment of cardiovascular disease

44 Contact information: Information about the research group: pages/cerg-cardiac- Exercise-Research- Group/

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