biotechnology research bit 220 mccc chapter 10. terminology molecular biology/genetics: the study of...
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Biotechnology Research
BIT 220
MCCC
Chapter 10
Terminology
Molecular Biology/Genetics: the study of gene structure and function at the molecular level
Molecular Biotechnology: the ability to transfer specific units of genetic information from one organism to another
Recombinant DNA Technology= gene splicing= genetic engineering=gene transplantation= gene cloning=molecular cloning
Genetics• Science of Heredity
• Explains similarities and differences between organisms
• Two Branches– Classical (Mendelian)
• cells contain pairs of ‘factors’ which determine physical characteristics
• these factors segregate during meiosis, independently
– Molecular
DNA/RNA Molecular Basis of Life
Central Dogma
DNA RNA protein control
Transcription Translation
DNA - Deoxyribonucleic AcidRNA - Ribonucleic Acid
DNA DNA
Replication (mitosis,meiosis)
DNA Structure
Replication
Gene Expression
Mutation
PROPERTIES OF DNA
1.Replicate Genotypic function
2.Mutate – chemically change and transmit these changes to
future generations - if mutant in germ lineevolutionary function
3.Gene expressionDirect the synthesis of proteinsphenotypic function
1. Miescher DNA 1868 isolated DNA from pus cells (NUCLEIN)
2. Frederic Griffith 10.1 and 10.21928 ‘transforming principle’ in pneumococcus
3. Oswald Avery, Colin MacLeod, Maclyn McCarty 10.31944 DNA was the active substance in the heat-killed S strain extracts because DNases destroyed the activity while RNases and proteases did no.
4. Chargaffthe molar concentration of A = T and G = Cwhich led to the discovery of base pair complementarity.
5. Wilkins and Franklin 10.10X-ray crystallographs (diffraction patterns) of
DNA -Review DNA structure: Figures 10.6, 10.7, 10.8, 10.11, 10.12, 10.13, Table 10.3
6. Watson and Crickdeduced a two-stranded structure wrapped in a right-handed helix with the bases
internal, the phosphates external, and an internal repeating subunit separated by 0.34 nm
NucleotideSmallest unit of nucleic acid
Three components1. sugar2. phosphate3. nitrogenous base
Other Nomenclature
Nucleoside : SUGAR and BASE (NO PHOSPHATE)
BASES NUCLEOSIDES NUCLEOTIDES
Adenine Deoxyadenosine Deoxyadenosine 5’-triphosphateGuanine Deoxyguanosine “” (dATP)Cytosine Deoxycytidine “”Thymine Deoxythymidine “”
Double Helix2 strands wrapped around one anotherSPIRAL STAIRCASE
Allows replicationAllows permanence
Bases on one side of helix arecomplementary to other strand
What draws two strands together?1. Bases hydrogen bond2. Bases are hydrophobic
DNA Structure
A. Sugar-Phosphate Backbone
covalent bonds between S and Pphosphodiester linkages
B. StairsSugar covalently bound to baseBases hydrogen bound to each otherA::TC:::G
DNA Antiparallel Strands
One strand runs 3’C (OH of sugar) to 5’ C (phosphate);
The other strand 5’ to 3’
Alternate forms
A 11 bp per turn/ right-handed
B 10.4 base pairs per turn/ right handed FOUND IN VIVO - physiological DNA form
Z 12 bp per turn/left handed
SupercoilingIn vivo negatively supercoiled (underwound)
Introduced when one or both strands nicked and strands rotate around one another
Figures 10.14 and 10.15
Chromosome StructureComposed of DNA (RNA viruses) and proteinssingle piece of nucleic acid
Prokaryotic (bacteria and virus)single chromosome/ 3000 genesE coli condenses DNA into loops 10.16no intronsall single copy DNAmonoploid
Eukaryotic1000 times more DNA than bacterianoncoding regions introns30-85% single copymost diploid
Eukaryotic Chromosomes
Chromatina. DNAb. non-histone proteinsc. histone proteins: 10.23 and 10.24
DNA wraps around dimers of each H2a, H2b, H3, and H4 forming nucleosome
H1 sits outside nucleosome
DNA Compaction10.27
Level 1 Nucleosome
Level 2 supercoiling of nucleosomes
Level 3 Scaffold composed of non-histone proteinspacking during metaphase (most condensation)
Centromeres Figures 10.29 and 10.30interchangeable among chromosomes110-120 bp in length.
Telomeres Figure 10.31contain special repeated DNA sequences that enable the ends of the chromosomes to be replicated, inhibit their degradation by DNA degrading enzymes, and prevent fusion with other chromosomes.
In vertebrates, the TTAGGG repeat is highly conserved and in humans 500-3000 repeats occur in telomeres which gradually shorten with age.
Eukaryotic DNA contains excess DNA, up to 50% or more, that does not code for proteins and comprises families of highly repeated sequence
elements or repetitive DNA (satellite DNA)three classes of DNA
1. unique or single-copy 1-10 copies per haploid genome
genes that encode proteinsregulatory sequences
2. moderately repetitive 10-100,000 copies
transposable elements (jumping genes)rRNA
histone ribosomal genes
3. highly repetitive >100,000 copies
telomerescentromeres
unknown function
Types of repeats
LINEs - Long Interspersed Nuclear Elements
SINEs - Short Interspersed Nuclear Elements
LTRs - Long terminal repeats
DNA transposons (transposable elements)
Denaturation/Renaturation
D: Two strands of DNA separate (Melt)
R: Two strand hydrogen bond through complementary bases (Annealing)
The kinetics of these processes will indicate the amount of repetitive DNA
Higher the copy number; the faster the renaturation occurs
Hybridization
Radioactive-labelled sequencesNucleic Acid probe contains radioactive isotopethis isotope emits charged particles as it decaysthese charged particles captured on emulsion (film)exposed silver halides produce back spots
Fluorecently-labelled sequencesTech Sidelight
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