GIO: L6 02-12 EXPLORING THE STRUCTURE AND FUNCTION OF GENES AND GENOMES

WHAT IS CLONING?

• TO MAKE IDENTICAL COPIES

• FOR DNA (OR MOLECULAR) CLONING

• 4 BASIC PROCEDURES ARE NECESSARY…

  1) A METHOD FOR CUTTING DNA AT PRECISE LOCATIONS

  • RESTRICTION ENDONUCLEASES

  2) A METHOD FOR COVALENTLY LIGATING TWO DNA MOLECULES

  • DNA LIGASE

  3) A PROCEDURE FOR MOVING RECOMBINANT DNA FROM THE TEST TUBE INTO A HOST

  • TRANSFORMATION, INFECTION, ELECTROPORATION

  4) A MECHANISM OF CARRYING AND REPLICATING SEGMENTS OF DNA WITHIN A HOST ORGANISM

  • DNA VECTOR

WHAT CLEAVE DNA AT SPECIFIC SEQUENCES?

• RESTRICTION ENDONUCLEASES

WHAT DOES DNA LIGASE DO? (1)

• JOINING THE FRAGMENTS!

• THESE ENDS ARE COHESIVE (THESE OVERHANGS ARE COMPATIBLE)

• THESE ENDS ARE NOT COHESIVE (THESE ENDS ARE NOT COMPATIBLE)

WHAT DOES DNA LIGASE DO? (2)

A) JOINING OF TWO FRAGMENTS CUT BY THE SAME RESTRICTION NUCLEASE

B) JOINING OF TWO FRAGMENTS CUT BY DIFFERENT RESTRICTION NUCLEASES

WHAT ARE CLONING VECTORS USED IN E. COLI?

WHAT IS CREATING AND PROPAGATING RECOMBINANT DNA?

WHAT DO ISOLATING GENES OFTEN REQUIRE (1)?

• A DNA LIBRARY

• LIBRARY: A COLLECTION OF DNA FRAGMENTS, EACH CARRIED IN A VECTOR

• GENOMIC LIBRARY: CONTAINS GENOMIC DNA FRAGMENTS, ORGANISM-SPECIFIC, CELL-TYPE INDEPENDENT

• CDNA LIBRARY: CONTAINS DNA COPIES OF MRNA, ORGANISM-SPECIFIC, CELL-TYPE SPECIFIC

WHAT DO ISOLATING GENES OFTEN REQUIRE (2)?

• REQUIRE A DNA LIBRARY

  

WHAT DO ISOLATING GENES OFTEN REQUIRE (3)?

• REQUIRE A DNA LIBRARY

• REVERSE TRANSCRIPTASE IS AN RNA-DEPENDENT DNA POLYMERASE

• RNASE H IS A NUCLEASE THAT DEGRADES RNA IN AN RNA-DNA HYBRID

• CDNA IS A DOUBLE-STRAND DNA COPY FROM AN RNA TRANSCRIPT

• INSERT DNA FRAGMENTS INTO PLASMIDS USING DNA LIGASE

  —> INTRODUCE PLASMIDS INTO BACTERIA—> CDNA LIBRARY

HOW CAN DNA MOLECULES BE SEPARATED?

• USING GEL ELECTROPHORESIS

• GEL ELECTROPHORESIS SEPARATES DNA MOLECULES BY SIZE

WHAT CAN DNA MOLECULES UNDERGO?

• DENATURATION AND HYBRIDIZATION

• DNA DOUBLE HELICES—> DENATURATION TO SINGLE STRANDS (HYDROGEN BONDS BETWEEN NUCLEOTIDE PAIRS BROKEN)—> RENATURATION RESTORES DNA DOUBLE HELICES (NUCLEOTIDE PAIRS RE-FORMED)

HOW CAN DNA SEQUENCES BE AMPLIFIED?

• BY PCR (POLYMERASE CHAIN REACTION): AMPLIFICATION OF DNA SEGMENTS MAKES POSSIBLE THE DETECTION OF PATHOGENIC VIRUS OR BACTERIA, IDENTIFICATION OF INDIVIDUALS (DNA FINGERPRINTING), AND SEVERAL SCIENTIFIC RESEARCH INVOLVING DNA MANIPULATION

• PCR REQUIRES A HEAT-STABLE DNA POLYMERASE

WHAT CAN PCR BE USED FOR?

• CLOTHING, DIAGNOSTICS, FORENSIC SCIENCE

• DNA ANALYSIS BY SHORT TANDEM REPEATS (STR)

  

HOW IS PCR USED IN FORENSIC SCIENCE (1)?

STR=SHORT TANDEM REPEAT

• STR LOCI ARE USEFUL IN DNA TYPING

• MOST COMMON REPEATS ARE 4-NT LONG

• STR LOCI MOST USEFUL IN DNA TYPING ARE 4-50 REPEATS (16-200 TOTAL BPS)

• MORE THAN 20000 TETRANUCLEOTIDE STR REPEATS HAVE BEEN CHARACTERIZED

PCR HAS FACILITATED STR ANALYSIS

• AS LITTLE AS 1 NG OF DNA IS NECESSARY FOR ANALYSIS

• PRIMERS FLANKING THE STR LOCI ARE USED FOR AMPLIFICATION

• PCR PRODUCTS OF DIFFERENTIATING LENGTHS ARE GENERATED FOR EACH ALLELE

• USE OF MULTIPLE ALLELES GENERATES UNIQUE “FINGERPRINTS”

HOW IS PCR USED IN FORENSIC SCIENCE (2)?

• IN THE CASE HERE, INDIVIDUALS A AND C CAN BE ELIMINATED FROM INQUIRIES, WHILE B IS A CLEAR SUBJECT

• A SIMILAR APPROACH IS NOW USED ROUTINELY IN PATERNITY TESTING

• THE MORE LOCI EXAMINED, THE MORE CONFIDENT WE CAN BE ABOUT THE RESULTS. WHEN EXAMINING THE VARIABILITY AT 5-10 DIFFERENT STR LOCI, THE ODDS THAT TWO RANDOM INDIVIDUALS WOULD SHARE THE SAME FINGERPRINT BY CHANCE ARE APPROXIMATELY ONE IN 10 BILLION

WHAT IS DNA SEQUENCING?

• SANGER SEQUENCING (DIDEOXY SEQUENCING) IS THE MOST WIDELY USED METHOD FOR SEQUENCING DNA

• USES DIDEOXYRIBONUCLEOTIDES (DDNTPS) THAT TERMINATE SYNTHESIS

• THE DDNTPS ARE FLUORESCENTLY LABELED

• G, T, C, AND A DDNTPS, EACH CARRY A DIFFERENT FLUROPHORE

WHAT IS GENOME SEQUENCING?

• THE DRAFT SEQUENCE OF THE HUMAN GENOME WAS COMPLETED IN 2000, AND THE FINAL SEQUENCE WAS COMPLETED IN 2004

• AS OF 2021, THE GENOMES OF 3,278 ANIMAL SPECIES HAD BEEN SEQUENCED

WHAT IS STUDYING GENE FUNCTION?

• RNA-SEQ CAN BE USED TO ANALYZE THE GENE EXPRESSION PROFILES OF EVERY CELL IN AN ORGANISM

• IN SITU HYBRIDIZATION TO MRNAS REVEALS PATTERNS OF GENE EXPRESSION DURING DEVELOPMENT

WHAT DOES RIBOSOME PROFILING REVEAL?

• REVEALS WHICH MRNAS ARE TRANSLATED INTO PROTEINS

  

WHAT ARE REPORTER GENES?

• CAN BE USED TO DETERMINE THE PATTERN OF A GENE’S EXPRESSION

• GREEN FLUORESCENT PROTEIN (GFP) CAN BE USED TO IDENTIFY SPECIFIC CELLS IN A LIVING ANIMAL

WHAT ARE CAENORHABDITIS ELEGANS?

SCARLET FLORESCENT REPORTER

WHAT CAN RNA INTERFERENCE BE USED FOR?

• TO STUDY GENE FUNCTION

WHAT DOES RECOMBINANT GENE TECHNOLOGY ALLOW?

• IT ALLOWS GENE—>PROTEIN!

• LARGE AMOUNTS OF PROTEINS CAN BE PRODUCED FROM CDNAS

• THIS IS IMPORTANT FOR:

  • SCIENTIFIC STUDIES

  • MEDICAL APPLICATIONS

• SOME MEDICALLY USEFUL PROTEINS:

  • HORMONES (INSULIN)

  • GROWTH FACTORS

  • THERAPEUTIC ANTIBODIES

  • VACCINES

• STRUCTURAL AND BIOCHEMICAL ANALYSES TO DETERMINE 3-D CONFORMATION AND ACTIVITY

• MANIPULATE AND INTRODUCE ALTERED GENES INTO CELLS OR ORGANISMS TO STUDY FUNCTION

WHAT IS GENE EDITING USING CRISPR?

• CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS

• THE CRISPR SYSTEM CAN

  • GUIDE RNA

  • CAS9 NUCLEASE

  • BACTERIAL VIRAL DEFENSE SYSTEM

  • USED BY RESEARCHERS FOR GENE EDITING

    

WHO WON THE NOBEL PRIZE FOR CRISPR?

WHAT IS GENE EDITING USING CRISPR AGAIN C. ELEGANS?

WHAT IS GENE EDITING USING CRISPR (DIAGRAM 1)?

WHAT IS GENE EDITING USING CRISPR (DIAGRAM 2)?

WHAT IS GENE EDITING USING CRISPR (DIAGRAM 3)?