5 PCR is used in:forensic science e.g. genetic fingerprinting;medical research e.g. genetic screening to find alleles for genetic diseases;scientific research e.g. to investigate DNA of extinct species to determine their relationship with living organisms;The Human Genome Project.Its purpose is to amplify very small quantities of DNA producing, many identical copies of the selected section of DNA in a very short time.
6 methodHeat to 95oC to break hydrogen bonds between complementary bases and separate strandsCool to 40-60oC & add excess primerPrimers are short single stranded sequences of about 20 nucleotides which are complementary to bases in part of the DNA strand being copied.Cooling allows the primers to bind (anneal) to each DNA strand at specific complementary pointsPrimers stop the DNA strands rejoiningThey bracket the DNA to be copied by attaching to the end of the DNA sequence and shows DNA polymerase where to start copying. There are 2 different primers as there are 2 different ends of the DNADNA replication can only start with a double stranded region
7 Heat to 70oC. Add free DNA nucleotides & thermostable DNA polymerase Polymerase copies each strand , starting with the primers.The polymerase is thermostable allowing the process to occur rapidly at the higher temperatures required, without denaturing. The enzyme was originally isolated from a bacterium that lives in hot springs.TWO DNA molecules will be formed following PCR and are used as templates as the process is repeated. This allows millions of copies of the original DNA to be produced in a very short time.
8 HEAT 95oC HEAT 70oC Cool 40-60oC primer DNA polymerase primer Heat causes Hydrogen bonds between bases to break.Heat acting in same way as DNA polymerase during replication.
9 key requirement AVOID CONTAMINATION with any other DNA source as both the original & contaminated DNA will be amplifiedDouble Helix heated, unwinds9
10 The Polymerase Chain Reaction sumanisinc brill animation
11 that are used to identify specific DNA sequences genetic markerssections of DNAthat are used to identifyspecific DNA sequencesAllows for comparison of DNA sequences between different people
12 restriction endonuclease enzymes Enzymes that recognise & cut specific nucleotide sequences.Originally from bacteria that use RE enzymes to cut DNA of attacking bacteriophages.
13 produces sticky ends because G & A don’t lie opposite each other. e.g. EcoR1recognises the sequence GAATTC, cutting between the G & A of each strandproduces sticky ends because G & A don’t lie opposite each other.
15 e.g. Bam1recognises the sequence GGATCC, cutting between the G & G of each strandproduces sticky ends because G & G don’t lie opposite each other.…G G A T C C……C C T A G G…
16 e.g. Hae 111recognises the sequence GGCC, cutting between the G & C of each strandproduces blunt ends because G & C lie opposite each other, these are not as useful as sticky ends.…G G C C……C C G G…
17 Restriction enzymes will cut DNA into a number of fragments, depending upon the number of recognition sites in the sample of DNA.The size of the fragment depends on how far apart the recognition sites are.If DNA has 4 recognition sites, 5 fragment lengths of DNA are produced.Recognitionsite 1Recognitionsite 2Recognitionsite 3Recognitionsite 4DNA SAMPLE5 fragments
18 If two samples of DNA are identical, treatment with a particular restriction enzyme will produce the same number of DNA fragments, each of similar size.However, different individuals have different DNA sequences and therefore produce different fragments when a particular restriction enzyme is used to cut an equivalent section of DNA.The fragments produced are called RFLPs, RESTRICTION FRAGMENT LENGTH POLYMORPHISMS.
19 This allows for comparison or identification of DNA samples PERSON ARecognitionsite 1Recognitionsite 2Recognitionsite 3Recognitionsite 4DNA SAMPLE5 fragmentsPERSON BRecognitionsite 1Recognitionsite 2Recognitionsite 3Recognitionsite 4Recognitionsite 5DNA SAMPLE6 fragmentsThis allows for comparison or identification of DNA samples
20 Restriction Fragment Length Polymorphisms RFLPSpronounced RiFlipsRestriction Fragment Length Polymorphisms
21 reasons why different people have different DNA sequencesSNPsMRSs
22 Single Nucleotide Polymorphisms snpspronounced snipsSingle Nucleotide Polymorphisms
23 A SNP is …… a difference in a single nucleotide in a section of DNA It can give rise to different alleles for a geneegs include sickle cell anaemia (chromosome 11)
24 Sickle cell anaemia Thr Pro Glu Glu Glu ...ACT CCT GAG GAG GAG... Thr normal Hb chainAmino acid sequenceThrProGluGluGluDNA base sequence...ACTCCTGAGGAGGAG...codon number4567sickle cell Hb chainAmino acidsequenceThrProValValGluDNA base sequence...ACTCCTGTGGTGGAG...
26 Microsatellite Repeat Sequences MRSsMicrosatellite Repeat Sequences(Also called variable number tandem repeats – VNTRs).
27 Only approximately 2% DNA codes for proteins and 3% has a regulatory role. The remaining 95% has unknown function and is called non-coding DNA.Within this DNA there are many sections where a small number of bases are repeated many timese.g. ….ATCATCATCATCATCATCATC…
28 These sequences are called microsatellite repeat sequences or short tandem repeats. The number of repeats of these MRSs are unique to an individual.
29 Short sequences of single stranded DNA dna PROBEShort sequences of single stranded DNAwith known nucleotide and base sequence.May be fluorescent or radioactively labelled, to enable it to be located.Radioactive probes contain radioactive isotopes of nitrogen in their nucleotide structure.
30 The DNA is treated to separate the DNA strands (heat) Add probe – it base pairs with complementary DNA sequence.Detect the target sequence:Fluorescent probe use a laser scannerRadioactive probe use X-ray film
31 They are used to identify specific gene sequences . Many inherited diseases are caused by a single allele with a known base sequencesA probe complementary to the allele nucleotide sequence is used:If it binds the disease is presentIf it doesn’t bind it is not present.
32 Pre implantation genetic diagnosis PGDPre implantation genetic diagnosisThis is the use of DNA probes to detect defective genes in an embryo to reduce the chance of a hereditary disease in a family.
35 Measuring genetic variation allows the following questions to be answered: Does a particular gene differ from person to person? Are two persons closely related?Are certain alleles associated with an hereditary disease?How do genes vary within a population? How much genetic diversity is there within a particular species?How do genes differ from species to species? How can this be used to unravel the taxonomic relationship among species?
37 Genetic (dna) fingerprinting Extract DNA from a samplee.g. from a crime scene, often PCR required to amplify the DNA2. Restriction Enzymes cut the DNA into different sized fragmentsSpecific MRSs used as they can be measured more accurately than RFLPs. Primers used to flank the MRS region involved to ensure fragments only come from that region
38 3. DNA fragments separated using gel electrophoresis. DNA is placed into separate wells in agar gel.An electric current is applied across the length of the gel, with the negative electrode closest to the wells of DNA.DNA has a negative charge, so moves through the spaces in the gel towards the positive electrode.Smaller DNA fragments move further than larger fragments.4. DNA is heated to separate the DNA strands to make it single stranded.
39 The gel will dry up, so the DNA fragments are transferred to a nylon membrane (process called Southern blotting)Radioactive or fluorescent DNA probes are added to the membrane and will join to the complementary base sequences in the microsatellite region selected. The sample is washed to remove any probes that do not base pair.The labelled DNA is added to X-ray film or a laser scanner is used to detect the radioactive or fluorescent probes and a pattern of bars appear, revealing the position of the DNA fragments: a DNA fingerprint.Diagram p82