1	CLSC320 Principles of Immunology Diagnostic Laboratory Immunology Program for Clinical Laboratory Science Unit - 15 Molecular Techniques
2	Unit – 15 Guidelines Reading assignment: Pages 314 - 335 of textbook Learning objectives: Those listed on page 315 of textbook Key terms: Those listed on pages 315 & 316 of textbook
3	Chapter Outline Structure & Synthesis of DNA Isolation of DNA Nucleic Acid Assays e e
4	Structure of DNA - 1 “ double-stranded molecule with double helix configuration “ each strand consists of: U four building blocks: (deoxyribonucleoside triphosphates) ]dTTP ]dCTP ]dATP ]dGTP deoxyribose + quanine + triphosphate sugar base deoxyribose + Adenine + triphosphate deoxyribose + cytosine + triphosphate deoxyribose + thymine + triphosphate “ DNA molecules consist of highly compacted units called chromosomes to which proteins are attached “ the nucleus of each human cell has 23 pairs of chromosomes (46 chromosomes) and constitutes the human genome. “ DNA molecules consist of a 3^' and 5' end
5	Structure & Synthesis of DNA Structure of RNA “ single-stranded molecule “ less stable than DNA: “ Thymine is replaced by uracil “ Sugar is ribose instead of deoxyribose as in DNA Synthesis of DNA “ site of protein synthesis is the: cytoplasm “ genetic information contained in DNA must be transferred from nucleus to cytoplasm by RNA. Three types of RNA: tRNA mRNA rRNA transfer RNA messenger RNA ribosomal RNA
6	Steps in Protein Synthesis - 1 Steps involved in protein synthesis: “ Initial step “ DNA replication “ DNA transcription “ Translation Initial step: “ double helix unwinds “ DNA strands begin to separate from each other “ RNA polymerase forms a short RNA molecules formed which serves as primer
7	Steps in Protein Synthesis - 2 DNA replication: “ each single strand of separated DNA acts as a template for the formation of a new Adaughter@ strand of DNA “ DNA polymerase proceeds with DNA synthesis linking deoxyribonucleotides in the 5^' to 3^' positions creating two double-stranded DNA molecules according to sequence of template DNA transcription: “ one gene contains the amino acid sequence (exon) that codes for one protein. This exon only makes up a portion of the genome “ the remaining DNA sequences are non-coding (introns) or Ajunk DNA@
8	Steps in Protein Synthesis - 3 DNA transcription: (cont=d) when the exon is activated a copy is made in the form of mRNA through the process of transcription Translation: “ mRNA carries units of 3 bases (triplets) and are these triplets are called codons “ the codon specifies the order of amino acids thus the protein formed “ there are 20 amino acids used to make up proteins “ there are 4 nucleotides (A, G, T, C of DNA) (and U of RNA) which results in 64 different codon possibilities
9	Products Involved in DNA Synthesis - 1 DNA polymerase enzyme that can synthesize DNA only in the 5^' to 3^' direction “ produces one complete new strand complementary to template strand “ the other DNA strand can only be synthesized in short segments going from 3^' to 5^' and these segments are joined together by DNA ligase to form complete DNA strand Primer “ short RNA sequences used to synthesize DNA in 3^' to 5^' direction
10	Products Involved in DNA Synthesis - 2 Nucleotides “ building blocks of nucleic acid “ composed of: U sugar (deoxyribose or ribose) U phosphate group U purine (adenine and quanine) U pyrimidine (cytosine, thymine, and uracil) Polynucleotides “ many nucleotides joined together
11	DNA Extraction Methods Classic procedure - consists of four steps: U release of DNA from cell U extraction of DNA from solution U precipitation of DNA U stabilization of DNA
12	Release & Extraction of DNA U release of DNA from cell ]detergent added that produces hole in cell membrane ]protease enzyme digests cellular and nucleic proteins ]DNA released into solution U extraction of DNA from solution ] using organic solutions: ephenol-chloroform added to solution and centrifuged proteins & lipids extracted DNA
13	Extraction of DNA ] using non-organic solutions: eprotein precipitating agent added and tube centrifuged proteins in precipitate extracted DNA in supernatant
14	Precipitating & Stabilization of DNA Uprecipitation of DNA from solution ] extracted DNA strands are precipitated out of supernatant using sodium or ammonium acetate + cold absolute ethanol ] pellet at bottom of tube contains DNA Ustabilization of DNA ]DNA washed in 70% ethanol and resuspended in water or buffer
15	RNA Extraction Consists of three steps: U lysing of cells with : guanidine thiocyanate U precipitation of RNA using : isopropanol + ethanol U RNA pellet resuspended in diluent
16	Use of Restriction Enzymes Ubacterial restriction enzymes (called endonucleases) have the ability to recognize short DNA base pair sequences and cleave the covalent bonds between adjacent nucleotides at a specific recognition site resulting in 4-8 recognition sequences Uthese restriction enzymes are named for the bacteria that produces them: Eco RI = Hind III = Eco = Escherichia coli R = RY13 strain I = 1st nuclease to be isolated Hin = Haemophilus influenzae d = Rd strain III = 3rd nuclease to be isolated
17	Nucleic Acid Assays U Electrophoretic Separation of DNA U Hybridization assays E Southern blot - for DNA E Restriction Fragment Length Polymerization (RFLP) E Dot Blot Hybridization E Northern blot - for RNA E DNA chip technology E Fluorescent in situ hybridization (FISH) U Nucleic Acid Amplification assays E Target amplification E Signal amplification
18	Electrophoretic Separation of DNA gel electrophoresis can separate the restriction enzyme-digested DNA according to size of nucleotide sequence produces bands called DNA or RNA Aladders@ standards are run and unknown ladders compared to known standards ethidium bromide is used to stain the various bands
19	Hybridization Assays allow fragments of known nucleic acid composition (called a Aprobe@) to locate the matching (complementary) sequence in unknown sample the denatured (seperated) DNA strands can be reformed (annealed) to produce a double-stranded DNA when one of the DNA strands is labelled with a signal-emitting tag such as: radioisotopes: ³²P, ³⁵S, or ¹²⁵I enzymes: alkaline phosphatase or peroxidase biotin + avidin + enzyme biotin + avidin + fluorochrome
20	Types of Hybridization Assays - 1 Southern blot Restriction Fragment Length Polymerization (RFLP) Dot Blot Hybridization E used to measure DNA E combines electrophoretic separation with hybridization procedure E used to determine polymorphism of HLA alleles and identification of an abnormal gene E also called immunoblot E sample DNA immobilized in solid phase such as: cellulose or nylon membrane E allows for multiple samples/membrane
21	Types of Hybridization Assays - 2 Northern blot DNA chip techonology E RNA used as probe E used to investigate gene expression E rRNA used as target E biochips (biological chips or microassays) serve as a solid support for a variety of combinations of relatively short oligonucleotides (pieces of genetic sequences used as probes) E used to identify alterations in gene sequences E fluorescent tags used to identify when DNA matching has occurred
22	Target Amplification Methods DNA target amplification methods E Ligase chain reaction (LCR) E Nucleic acid sequence-based amplification (NASBA) E Transcription-mediated amplification (TMA) E Polymerase chain reaction (PCR) Goal of methods: E increase concentration of target nucleic acid E amplify the probe
23	Ligase Chain Reaction (LCR) & Polymerase Chain Reaction (PCR) Ligase chain reaction (LCR) etarget sequence must be completely known vs PCR where it is not known eadvantage over PCR : ]able to detect point mutations Polymerase chain reaction (PCR) eclinical applications of PCR: ]diagnosis of sickle cell disease ]oncology evaluation ]genetics studies ]infectious diseases (NAT) ]forensic medicine (O.J. Simpson)
24	NASBA Method eNucleic Acid Sequence-Based Amplification (NASBA) E used to amplify target RNA E employs three enzymes: ]reverse transcriptase: ]ribonuclease H: ]T7 RNA polymerase: -makes complementary DNA strand from RNA and it serves as template for rurther replication -degrades RNA to allow for a complete-double stranded DNA -produces multiple copies of RNA from DNA template clinical application: detection of viral RNA
25	Signal Amplification Methods Developed to increase signal strength by increasing concentration of the label Methods of signal amplification include: euse of multiple enzymes euse of multiple probes euse of two-tiered probes euse of multiple probes & multiple enzymes
26	OSF - Molecular Fingerprinting On page 323AOne Step Further@ presents a more in-depth discussion of Molecular Fingerprinting. This presentation is contained on a separate slide presentation called A One Step Further #17" The student may call up the slide program OSF-17 later or click on the arrow below to view slides now.
27	OSF – AIDS Testing On page 329AOne Step Further@ presents a more in-depth discussion of testing for AIDS. This presentation is contained on a separate slide presentation called A One Step Further #18" The student may call up the slide program OSF-18 later or click on the arrow below to view slides now.
28	Molecular Techniques - 27 Press the ESC key to end program
29	One Step Further Molecular Fingerprinting OSF - 17 Page 323 7click arrow to return to main program
30	Molecular Fingerprinting - 2 Application: etechniques used for the detection of polymorphism of HLA alleles Molecular techniques: eRestriction fragment length polymorphism (RFLP) esequence-specific priming (SSP) esequence-specific oligonucleotide probing (SSOP) esequence-base typing (SBT) uses Southern blotting technique identifies HLA loci that code for individual HLA antigens alternative name = Amolecular fingerprinting@ uses PCR/gel electrophoresis technique detects polymorphism at allele level uses PCR/hybridization probes to PCR products detects HLA alleles uses PCR/nucleotide sequencing of PCR products detects alleles at exact sequence level
31	Molecular Fingerprinting - 3 Restriction fragment length polymorphism (RFLP) technique: eDNA is extracted from patient=s cells eDNA is visualized using Southern blotting technique eHLA iantigens are identified by comparing patient=s band patterns to control (known) band patterns ecan be used to distinquish one individual from another
32	Molecular Fingerprinting - 4 7click arrow to return to main program
33	One Step Further Testing for AIDS OSF - 18 Pages 329 & 330 7click arrow to return to main program
34	Testing for AIDS - 2 Definition of AIDS: eAcquired Immunodeficiency Syndrome Causative agent: eHuman Immunodeficiency Virus-1 (HIV-1) Characteristics of HIV-1: eit is a retrovirus eit contains two strands of RNA ealso contains several enzymes eit=s capsid contains lipids egp120 antigen of virus attaches to CD4 antigen on cells ethree major genes and their functions: Ženv gene: Žgag gene: Žpol gene: Codes for glycoproteins in outer envelope (gp120 & gp41) Codes for core proteins (p55, p40 & p24) Codes for enzymes reverse transcriptase (p66) & (p51), protease (p11), & integrase (p51)
35	Testing for AIDS - 3 Clinical course of HIV infection: ePrimary HIV infection eClinical latency eClinical AIDS Žhigh levels of HIV-1 detected in peripheral mononuclear cells and plasma Žanti-HIV-1 antibodies appear in plasma Žlevels of HIV-1 viral load desreases Ždiffers from individual to individual Žmay be rapid transition to clinical AIDS Žmay be 10 years or more for transition to clinical AIDS Žviral replication occurs in lymphoid tissues Žsmall amount of HIV-1 in peripheral blood Žproduction of HIV-1 = destruction of HIV-1
36	Testing for AIDS - 4 Clinical course of HIV infection: ePrimary HIV infection eClinical latency eClinical AIDS Žwhen immune system destruction of HIV-1 fails to keep up with production of HIV-1 Žsevere depression of immune system due to destruction of CD4 lymphocytes Žopportunistic infections and neoplasms develop: wPneumocystis carnii wKarposi=s sarcoma
37	Testing for AIDS - 5 Detection and Diagnosis of HIV-1 infections: eAntibody detection eAntigen detection ŽELISA test used as screening test ŽWestern blot test used as confirmatory test to detect: wanti-p24 wanti-p31 wanti-p41 2 out of 3 must be present for a positive diagnosis ŽQualitative detection ŽQuatification of HIV wuses amplification of HIV DNA by PCR wused to detect HIV-1 in newborns prior to Ab=s being produced wuses NASBA to establish viral load wused to monitor patient progress or threat to fetus
38	Testing for AIDS - 6 7click arrow to return to main program