Unit 6 DNA

Deoxyribonucleic acid – (DNA) – the molecules that carry the body’s genetic information Deoxyribonucleic acid – (DNA) – the molecules that carry the body’s genetic information Chromosomes – threadlike structure in the cell nucleus composed of DNA, where genes are located Chromosomes – threadlike structure in the cell nucleus composed of DNA, where genes are located Gene – fundamental unit of heredity Gene – fundamental unit of heredity Instruct the body’s cells to make proteins which determine all traits Instruct the body’s cells to make proteins which determine all traits Each gene is made of DNA that is designed to carry out a body function Each gene is made of DNA that is designed to carry out a body function DNA

DNA Structure Discovered by Watson and Crick in 1950s Discovered by Watson and Crick in 1950s Polymer – composed of repeating units or monomers Polymer – composed of repeating units or monomers –Nucleotide – make up DNA Made of: Made of: –A sugar –A phosphate group –A nitrogenous base (A, G, C, T)

Sugars and phosphates make up the backbone and nitrogenous bases connect in the middle. Sugars and phosphates make up the backbone and nitrogenous bases connect in the middle. DNA can be DNA can be millions of base pairs long

DNA Shape DNA is made of two strands twisted into a double helix DNA is made of two strands twisted into a double helix A connects with T A connects with T G connects with C G connects with C –This is known as complementary base pairing

Write the complementary strand for the following strand of DNA Write the complementary strand for the following strand of DNATAACGGAATTAAATTTCTGCCGTAAACCCCG

Life Instructions DNA is a book of instructions DNA is a book of instructions A, T, C, and G are like the alphabet used to create the book A, T, C, and G are like the alphabet used to create the book The order these letters are arranged in is unique to each person and defines the role and function of a DNA molecule. The order these letters are arranged in is unique to each person and defines the role and function of a DNA molecule.

DNA and Proteins DNA codes for amino acids DNA codes for amino acids Three letters in a row in DNA called a codon code for a specific amino acid Three letters in a row in DNA called a codon code for a specific amino acid

The order of amino acids determines a proteins shape and function The order of amino acids determines a proteins shape and function Proteins carry out all the major functions in living things Proteins carry out all the major functions in living things Example: Hemoglobin carries oxygen on red blood cells Example: Hemoglobin carries oxygen on red blood cells –CCT – GAG – GAG Normal hemoglobin –CCT – GTG – GAG Sickle Cell Anemia

DNA Replication Making new DNA from existing DNA Making new DNA from existing DNA The two strands unwind or uncoil The two strands unwind or uncoil Each strand is then open to attach to new nucleotides Each strand is then open to attach to new nucleotides The strands are exposed to free nucleotides which attach to the open strands according to base pairing rules The strands are exposed to free nucleotides which attach to the open strands according to base pairing rules –A with T –G with C Makes two identical copies of DNA Makes two identical copies of DNA

DNA Polymerase An enzyme that brings nucleotides in the correct sequence according to the original strand An enzyme that brings nucleotides in the correct sequence according to the original strand Proofreads the DNA strand for any mismatched base pairs and replaces the incorrect base with the correct one Proofreads the DNA strand for any mismatched base pairs and replaces the incorrect base with the correct one

DNA Typing with Tandem Repeats

Tandem Repeat A section of DNA that codes for nothing A section of DNA that codes for nothing Found between coding sections of DNA that code for amino acids or proteins Found between coding sections of DNA that code for amino acids or proteins They do not affect our outward appearance or control any genetic function but they are unique to us They do not affect our outward appearance or control any genetic function but they are unique to us We inherit them from our parents We inherit them from our parents Can distinguish one person from another through DNA typing Can distinguish one person from another through DNA typing

Restriction Fragment Length Polymorphism (RFLP) DNA is cut with a restriction enzyme that acts like a pair of scissors cutting the DNA at a specific point DNA is cut with a restriction enzyme that acts like a pair of scissors cutting the DNA at a specific point Different lengths of DNA base pairs are made from this restriction enzyme Different lengths of DNA base pairs are made from this restriction enzyme Each person has a different amount and length of DNA when it is cut with the restriction enzyme based on what they inherited from their parents Each person has a different amount and length of DNA when it is cut with the restriction enzyme based on what they inherited from their parents The length differences associated with the DNA strand can be used to distinguish one person from another The length differences associated with the DNA strand can be used to distinguish one person from another

Electrophoresis After DNA molecules are cut up by restriction enzymes they are separated by electrophoresis After DNA molecules are cut up by restriction enzymes they are separated by electrophoresis DNA is placed on a plate coated with a gel medium DNA is placed on a plate coated with a gel medium It is subjected to an electrical charge and DNA fragments are pulled toward the positive end of the gel because it is slightly negative It is subjected to an electrical charge and DNA fragments are pulled toward the positive end of the gel because it is slightly negative Smaller DNA fragments move faster along the plate than larger ones so the fragments are separated according to size Smaller DNA fragments move faster along the plate than larger ones so the fragments are separated according to size

Hybridization The process of joining two complementary strands of DNA to form a double stranded molecule The process of joining two complementary strands of DNA to form a double stranded molecule Fragments are transferred to a nylon membrane this is called Southern blotting Fragments are transferred to a nylon membrane this is called Southern blotting The sheet is treated with radioactive labeled probes complimentary to the RFLP being identified so we can see it The sheet is treated with radioactive labeled probes complimentary to the RFLP being identified so we can see it

DNA Typing with RFLP Nylon sheet is placed against X-ray film and exposed for several days Nylon sheet is placed against X-ray film and exposed for several days Film is processed and bands appear where the radioactive probes stuck to the fragments on the nylon sheet Film is processed and bands appear where the radioactive probes stuck to the fragments on the nylon sheet The length of each fragment is determined by running known DNA samples with the unknown samples The length of each fragment is determined by running known DNA samples with the unknown samples You look to match the band sets between the known and unknown DNA You look to match the band sets between the known and unknown DNA RFLP has been replaced in recent years by PCR technology RFLP has been replaced in recent years by PCR technology

PCR Replaced RFLP in mid-1990s Replaced RFLP in mid-1990s Uses DNA polymerase to replicate or Uses DNA polymerase to replicate or amplify a strand of DNA millions of times Short sequences of DNA on each side of the region you want to copy must be identified Short sequences of DNA on each side of the region you want to copy must be identified –Primer – a short strand of DNA used to target a region of DNA for replication by PCR

Steps of PCR 1. Heat DNA to 94 degrees to separate the DNA strands 2. Add Primers to the strands and they attach to the surrounding sequences 3. Add DNA polymerase and a mixture of free nucleotides 1. DNA polymerase directs the rebuilding of the DNA by adding the appropriate bases one at a time 4. Final product is two new DNA segments 1. Usually cycles are performed to make more than one billion copies of DNA 2. Each cycle takes less then 2 minutes

Advantages of PCR DNA does not have to be long DNA does not have to be long Short DNA is less likely to degrade Short DNA is less likely to degrade Can extract DNA more easily from items found at the crime scene like: Can extract DNA more easily from items found at the crime scene like: Envelopes Envelopes Stamps Stamps Soda cans Soda cans Cigarette butts Cigarette butts

Short Tandem Repeats (STRs) Most successful and widely used DNA profiling procedure Most successful and widely used DNA profiling procedure STR – locations (loci) on the chromosome that contain three to seven repeating base pairs STR – locations (loci) on the chromosome that contain three to seven repeating base pairs It is a short strand of DNA so it is less likely to degrade and can often be collected from areas where extreme decomposition has occurred. It is a short strand of DNA so it is less likely to degrade and can often be collected from areas where extreme decomposition has occurred. Ideal to use with PCR because they are a small amount of DNA so PCR can make many copies Ideal to use with PCR because they are a small amount of DNA so PCR can make many copies

TH01 A commonly used STR A commonly used STR Repeating segment A-A-T-G Repeating segment A-A-T-G Seven variations of TH01 have been found Seven variations of TH01 have been found –Each contains 5-11 repeats of A-A-T-G TH01 is extracted from biological material and copied using PCR TH01 is extracted from biological material and copied using PCR Separated by electrophoresis Separated by electrophoresis Distance traveled on the gel can determine the number of repeats in the STR Distance traveled on the gel can determine the number of repeats in the STR

Every person has 2 STRs one from each parent Every person has 2 STRs one from each parent So the total number of repeats can help you to identify the person So the total number of repeats can help you to identify the person

Multiplexing Hundreds of different types of STRs are found in human genes Hundreds of different types of STRs are found in human genes The more STRs you can find in someone’s DNA the smaller the percentage of the population from which it could of come from The more STRs you can find in someone’s DNA the smaller the percentage of the population from which it could of come from Multiplexing – using PCR to look for more than one STR at a time Multiplexing – using PCR to look for more than one STR at a time

STR blue kit detects three STRs: D3S1358, vWA, and FGA STR blue kit detects three STRs: D3S1358, vWA, and FGA The size of all three segments is different and easily seen on a gel The size of all three segments is different and easily seen on a gel US has 13 known STRs which they use in CODIS US has 13 known STRs which they use in CODIS When trying to find an STR you must know the sequence of the STR and the sequences of the bases around it When trying to find an STR you must know the sequence of the STR and the sequences of the bases around it This allows us to use primers to copy the correct sections of DNA using PCR This allows us to use primers to copy the correct sections of DNA using PCR

DNA Typing with STRs Open your book to page 328 Open your book to page 328 This table list the 13 STRs used in CODIS and their probability of identity This table list the 13 STRs used in CODIS and their probability of identity This is a measure of the likelihood that 2 individuals selected at random will have an identical STR type This is a measure of the likelihood that 2 individuals selected at random will have an identical STR type It is better to multiplex or look for many STRs because this helps to individualize samples as being from one person. It is better to multiplex or look for many STRs because this helps to individualize samples as being from one person. Each person should have a different combination of STRs so the more STRs that are identified in a DNA sample the more likely you are to connect it to one person. Each person should have a different combination of STRs so the more STRs that are identified in a DNA sample the more likely you are to connect it to one person. Example: A combination of the first 3 STRs in the table occurs in 1 in 5,000 people Example: A combination of the first 3 STRs in the table occurs in 1 in 5,000 people A combination of the first 6 STRs occurs in 1 in 2 million for the Caucasian population A combination of the first 6 STRs occurs in 1 in 2 million for the Caucasian population If the top 9 STRs are found in combination this occurs in 1 in one billion people If the top 9 STRs are found in combination this occurs in 1 in one billion people The combination of all 13 STRs occurs in 1 in 575 trillion Caucasians and 1 in 900 trillion African-Americans. The combination of all 13 STRs occurs in 1 in 575 trillion Caucasians and 1 in 900 trillion African-Americans.

Capillary Electrophoresis Used instead of regular gel electrophoresis because: Used instead of regular gel electrophoresis because: It reduces analysis time It reduces analysis time Automates sampling and data collection Automates sampling and data collection Carried on a thin glass column Carried on a thin glass column Each end of the column is immersed in reservoir buffer liquid that holds electrodes to supply high energy voltage Each end of the column is immersed in reservoir buffer liquid that holds electrodes to supply high energy voltage Column is coated with a gel polymer Column is coated with a gel polymer DNA sample is injected using a syringe into the column DNA sample is injected using a syringe into the column STR fragments move through the column because they are attracted to the electrical charges STR fragments move through the column because they are attracted to the electrical charges They move according to size They move according to size As each DNA segment passes through the end of the column they are recorded by a detector and the results are displayed by an electropherogram. As each DNA segment passes through the end of the column they are recorded by a detector and the results are displayed by an electropherogram.

Sex Identification with STR Amelogenin gene – located on the x and y chromosome Amelogenin gene – located on the x and y chromosome –Shorter by 6 bases pairs in the X than in the Y –Electrophoresis separates the DNA by size after PCR –Males have two bands because X and Y are different sizes –Females have one band because both X chromosomes are the same size

Y-STRs More then 20 different Y-STRs found just on the Y chromosome More then 20 different Y-STRs found just on the Y chromosome Seventeen have been characterized and can be used to separate one man’s DNA from another Seventeen have been characterized and can be used to separate one man’s DNA from another Example: more then one man is involved in a sexual assualt Example: more then one man is involved in a sexual assualt

Mitochondrial DNA Found outside the nucleus Found outside the nucleus Inherited from the mother only Inherited from the mother only Mitochondria provide energy in the cell Mitochondria provide energy in the cell Each cell has hundreds to thousands of mitochondria Each cell has hundreds to thousands of mitochondria Each mitochondria has several loops of DNA Each mitochondria has several loops of DNA So there are hundreds to thousands of copies of mitochondrial DNA in each cell So there are hundreds to thousands of copies of mitochondrial DNA in each cell

Mitochondrial DNA is used when: Nuclear DNA is degraded Nuclear DNA is degraded –Charred remains Nuclear DNA is present in small amounts Nuclear DNA is present in small amounts –Hair shaft All children from the same mother will have the same mtDNA All children from the same mother will have the same mtDNA –You cannot used mtDNA to tell one from the other

mtDNA Downfalls The test is harder to run then nuclear DNA The test is harder to run then nuclear DNA It is more time consuming then nuclear DNA profiling It is more time consuming then nuclear DNA profiling It costs more money then nuclear DNA profiling It costs more money then nuclear DNA profiling Only a handful of public and private labs receive mtDNA evidence Only a handful of public and private labs receive mtDNA evidence FBI limits the types of cases it used mtDNA testing on FBI limits the types of cases it used mtDNA testing on

Composition of mtDNA Made in a circular loop Made in a circular loop Each loop contains enough A, T, C, and G to make up thirty-seven genes involved in making energy in the mitochondria Each loop contains enough A, T, C, and G to make up thirty-seven genes involved in making energy in the mitochondria 2 regions are highly variable in humans 2 regions are highly variable in humans –HV1 – hypervariable region one –HV2 – hypervariable region two

Hypervariable regions Analyzing these regions is tedious Analyzing these regions is tedious PCR is used to make copies of each region PCR is used to make copies of each region The order of A, C, T, and G is then determined The order of A, C, T, and G is then determined FBI and Armed forces DNA Identification Laboratory have collaborated to compile an mtDNA database containing the base sequences for HV1 and HV2 FBI and Armed forces DNA Identification Laboratory have collaborated to compile an mtDNA database containing the base sequences for HV1 and HV2 Once HV1 and HV2 in a case are discovered they are inserted into the database to see how common or rare they are Once HV1 and HV2 in a case are discovered they are inserted into the database to see how common or rare they are Many of these sequences are unique to people but they are not as accurate as STRs in nuclear DNA so mtDNA should only be used when nuclear DNA is not available Many of these sequences are unique to people but they are not as accurate as STRs in nuclear DNA so mtDNA should only be used when nuclear DNA is not available

Mitochondrial DNA is used in Court First case to use mtDNA was in 1996 in Tennessee First case to use mtDNA was in 1996 in Tennessee –State of Tennessee v. Paul Ware Used to link two hairs discovered at the crime scene to the defendant Used to link two hairs discovered at the crime scene to the defendant There was no blood and semen evidence There was no blood and semen evidence mtDNA is often used to identify skeletal remains as well mtDNA is often used to identify skeletal remains as well –Abundant amount of mtDNA found in skeletal remains –Used to identify soldiers in unmarked graves in Vietnam –Can compare to anyone who has the same mother, grandmother, etc.

Collection and Preservation of DNA Evidence Only 18 cells that have DNA are needed for an STR profile Only 18 cells that have DNA are needed for an STR profile –An amount of DNA lower then 18 is known as the low copy number Advances in technology have allowed us to extract an STR profile from only 9 cells Advances in technology have allowed us to extract an STR profile from only 9 cells –Can extract DNA from envelopes, cups, chewing gum, sweat band of a hat, bed sheet containing dead skin or epithelial cells –The hope is to one day be able to extract DNA from a single cell –See chart on page 339

Collection of Biological Evidence Recorded using notes, sketches, and photographs Recorded using notes, sketches, and photographs If blood spatter is present that should be examined first to try and reconstruct events that may have taken place If blood spatter is present that should be examined first to try and reconstruct events that may have taken place Wear gloves to prevent contamination and to protect yourself from disease Wear gloves to prevent contamination and to protect yourself from disease –Changed frequently during collection

Biological Evidence Collection Continued.... May require a face mask, shoe covers, and coveralls May require a face mask, shoe covers, and coveralls All clothing from victim and suspect must be collected so a possible link can be established between the two people All clothing from victim and suspect must be collected so a possible link can be established between the two people Investigators should look for towels, rags, or hankerchiefs that may have been used to clean up blood Investigators should look for towels, rags, or hankerchiefs that may have been used to clean up blood Cracks and crevices in the floor should be examined for blood evidence Cracks and crevices in the floor should be examined for blood evidence

Packaging Biological Evidence No plastic or air tight containers No plastic or air tight containers –Moisture causes bacteria and fungi to grow Use paper bag or well ventilated box Use paper bag or well ventilated box Entire item should be submitted for evidence Entire item should be submitted for evidence –If not possible dried blood is best removed using a sterile cotton- tipped swab moistened with distilled water –Swabs must air dry for 5-10 minutes, should not be packaged wet –Portion of the unstained surface is swabbed as a substrate control Evidence should be refrigerated until it is brought to the lab Evidence should be refrigerated until it is brought to the lab –Exception: blood and soil must be frozen

Obtaining DNA Reference Specimens 7 ML of blood should be extracted from possible suspects and victims 7 ML of blood should be extracted from possible suspects and victims –Collected in a sterile vacuum tube –Contains EDTA: preserves DNA and inhibits enzymes that degrade DNA –Kept refrigerated until they reach the lab Buccal swab – cotton swab is used to rub the inside of the subject’s cheek Buccal swab – cotton swab is used to rub the inside of the subject’s cheek If individual is not available or does not want to give a reference sample DNA can be collected from: If individual is not available or does not want to give a reference sample DNA can be collected from: –Toothbrush, comb, hairbrush, razor, soiled laundry, used cigarette butts, earplugs, etc.

Contamination of DNA Evidence Can occur by: Can occur by: –Introduction of foreign DNA by coughing or sneezing onto a stain during the collection process –Transfer of DNA when items are packaged incorrectly next to each other –Can tell with STRs because there should be a two band pattern. If there are more then two bands that suggest contamination

To Minimize Contamination of DNA Evidence You Should: Change gloves before handling new pieces of evidence Change gloves before handling new pieces of evidence Collect a substrate control for possible subsequent laboratory examination Collect a substrate control for possible subsequent laboratory examination Pick up small items of evidence such as cigarette butts and stamps with clean forceps (disposable forceps are used once and thrown away) Pick up small items of evidence such as cigarette butts and stamps with clean forceps (disposable forceps are used once and thrown away) Always package each item of evidence in its own well-ventilated container Always package each item of evidence in its own well-ventilated container