Law: DNA replication and Biotechnology

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Griffith's Experiments (1928)

1. used two strains of Streptococcus pneumoniae and injected them into mice.
2. live injected S strain killed mice and live injected R strain did not kill mice
3. heat killed S strain injected into did not kill mice
4. mixture of heat killed S strain and R strain killed some mice (found presence of live S strain in blood)
5. genetic material was passed from heat killed S strain to R strain to make more virulent.

2 Types of Strains

1. S strain = virulent strain that leads to death in mice, looked smooth and shiny (capsule that allowed bacteria to trick mice immune systems)
2. R strain= non-virulent, mice didn't get sick, looked rough

Transformation

1. process of transforming genetic material between cells by DNA molecules.

Transforming Factor

1. the molecular agent of transformation, DNA.

J.L. Alloway Experiments (1933)

1. similar experiment as Griffith's but no mice
2. in test tube, he incubated mixture of heat killed S strain with Live R strain bacteria = all R strain bacteria turned into S strain bacteria

Oswald Avery (1944)

1. discovered transforming factor was DNA.
2. purified chemical components of S cells into carbohydrates, fats, proteins, and nucleic acids.
3. each component was mixed with live R cells and injected into mice
4. Only the mice injected with S cell nucleic acids and R cells died of pneumonia.

Conclusions of Oswald Avery Experiments

1. DNA is genetic material
2. DNa controls the synthesis of specific products, ie) S strain capsule gone to R strain.

Alfred Hershey and Martha Chase (1940's-50's)

1. bacterial phage was used to infect E.coli.
2. phage contained a simple structure: protein coat and DNA which made it easy to detect what genetic material.
3. Performed 2 experiments

Experiment #1

1. injected radioactive sulfur to phage - coat proteins
2. allowed phage to infect bacteria -centrifuged to get bacteria into pellet.
3. checked phage in bacteria for radioactivity
4. Viral protein coats were radioactive
5. bacterial with viral genetic info. was non-radioactive

Experiment #2

1. injected radioactive phosphorus to phage - coat nucleic Acids
2. allowed phage to infect bacteria - centrifuged to get bacteria into pellet
3. checked media and bacteria for radioactivity
4. found the bacteria with viral genetic information was radioactive.
5. Therefore, genetic material was DNA enters bacterial cells and replicates.

DNA nucleotides

1. smaller sub-units of DNA
2. deoxyribose = pentose sugar (C'2 has no OH group)
3. phosphate group = PO₄⁻ (strongly acidic)
4. nitrogenous base = purines (A and G), pyrimidines (T and C)

Purines

1. double ring structure
2. Adenine
3. guanine

Pyrimidines

1. single ring structure
2. Thymine
3. Cytosine
4. Uracil (only in RNA)

DeoxynucleoSIDES

1. contains N base and pentose sugar only (NO phosphate group)
2. A- deoxyadenosine
3. T- Thymidine
4. G- deoxyguanosine
5. C - deoxycytidine

DeoxynucleoTIDES

1. base + pentose sugar + phosphate
2. A (dAMP) = deoxyadenosine monophosphate
3. T (TMP) = thymidine monophosphate
4. G (dGMP) = deoxyguanosine monophosphate
5. C (dCMP) = deoxycytidine monophosphate

DNA POLYnucleotides

1. linked nucleotides together by phosphate diester linkage
2. forms long chain of linked nucleotides
3. linkage occurs at C'3 with OH ↔O on (PO₄⁻) of C'5

DNA Characteristics known before structure was known

1. amt. of DNA varies in cells of different species
2. amt. of DNA is constant in cells of dame species
3. gametes have 1/2 DNA of other body cells
4. Chargaff's Rule: concentrations of T = A and concentrations of C= G

Watson and Crick

1. worked on the structure of DNA by sifting and organizing info. that was already available about DNA.
2. they focused on X-ray diffraction studies which provided clues to helical structure of DNA
3. chemical analysis of DNA base composition from many different organisms

Wilkins and Franklin

1. studied X ray diffraction from highly purified DNA samples
2. indicated that DNA had a helical shape with a constant diameter, PO₄⁻ were on the outside of helix, and bases were stacked inside

Linus Pauling

1. explained to Watson and Crick Herman Paulings discovery that DNA contained a double helix
2. linked Watson and Crick to an organic chemist who was able to tell them that N bases H bonded with each other in their ketone form.

DNA structure characteristics

1. DNA is composed of 2 polynuecleotide chains running in opposite directions - antiparallel
2. 2 polynucleotide chains are coiled to form a double helix (spiral staircase)
3. in each chain, sugar and phosphate are on the outside of the molecule
4. T double bonded with A, and C triple bonded with G (H bonds)

3 Properties of Watson and Crick Model

1. genes are stored in sequence of bases, 4ⁿ possibilities, n= # of nucleotides
2. offers molecular explanation for mutations, changes in # or order = mutation
3. complementary nature of two polynucleotide chain helps explain how DNA is copied. (semi-conservative)

Base stacking distance

.34nm
(vertical distance between base pairs)

Length of complete turn of double helix

1. 3.4nm (10bp)

Diameter of Double Helix

1. 2.0nm

RNA structure

1. ribose sugar
2. A-U, C-G
3. PO₄⁻
4. usually single stranded

Functions of RNA

1. transfers genetic info. from nucleus to cytoplasm
2. synthesizes proteins
3. components of ribosomes

Meselson-Stahl experiment

1. grow bacteria in heavy isotope of Nitrogen (¹⁵N)
2. transfer bacteria to normal nitrogen media (¹⁴N)
3. sample bacteria after one replication - isolate DNA, centrifuged DNA and separate by density
4. sample bacteria after 2nd replication - looked to see how many bands appeared
5. supports that DNA undergoes semi-conservative replication

Results of Meselson-Stahl Experiment

1. After 0 minutes (parental generation) - there was only one strand of DNA
2. After 20 min. (first generation) - there were 2 bands
3. After 40 min. (2nd generation) - there were 4 bands, two with original DNA and two with first generation DNA

Semiconservative Replication

1. occurs in the nucleus
2. double helix unwinds
3. parental strands act as templates for new strands
4. complementary base pairing is utilized
5. parent and daughter strands rewind into duplex
6. DNA is copied exactly

Initiation of DNA replication

1. begins at origin of replication - there are multiple sites through out the chromosomes
2. DNA helicase unwinds double helix
3. replication bubbles form - replication will occur in both directions

Characteristics of Replication bubbles

1. bubbles expand and eventually merge
2. chromosomes are replicated rapidly

Replication Fork

1. area of wound and unwound DNA duplex
2. replicating is taking place in this region

Process of DNA synthesis

1. single stranded binding proteins (SSB) bind at replication fork - hold replication fork open
2. DNA polymerase III bind to each unwound single DNA strand - making new DNA strands and proof reading for mutations.

DNA polymerase III

1. grabs nucleotides from pools in the nucleus
2. grabs a dNTP - nucleotide with 3 phosphates
3. polymerase breaks diphosphate for bonding to occur
4. adds complentary dNTPs in correct order using template (parent) strand (all 4 deoxynucleotide triphosphates are present)

Rules of DNA synthesis

1. DNA polymerase III can only add nucleotides onto the 3' end of chain (free 3' OH groups)
2. daughter DNA is synthesized 5' to 3'
3. diphosphates are clipped
4. nucleotides are linked together by phosphatediester linkage

Leading Strand

1. daughter strand that is synthesized in 5'-3' direction
2. parent strand is in 3'-5' direction

Lagging Strand

1. daughter strand that is synthesized in 3'-5' direction
2. parent strand is in 5' - 3' direction

Synthesis of Leading Strand

1. starts at origin of replication
2. daughter strand moves in direction of replication fork (elongates in 5'-3' direction)
3. fork continues to unwind complex
4. new continuous strand follows unwinding fork

Problems with Lagging Strand synthesis

1. cannot follow replication fork
2. DNA polymerase III can only make DNA 5'-3' direction
3. Replication fork is moving in wrong direction for this strand
4. polymerase must move in opposite direction as fork

Okazaki Fragments

1. this strand must be made in small pieces (5'-3') direction
2. DNA polymerase III moved away from replication fork
3. DNA is made in the reverse direction of leading strand
4. DNA ligase will join fragments together

DNA Priming

1. RNA primase is the enzyme that makes a small piece of RNA with a free 3'-OH (denovo) - makes an RNA primer that complements base pairs
2. RNA primase falls off chain once primer is made
3. DNA polymerase III can now use the 3' OH end of this primer to begin making DNA

RNA Primer

1. is only made once for leading strand but has to be made many times for the lagging strand
2. 1 primer for each okazaki fragment

DNA Polymerase I

removes RNA primer and fills in the gap with DNA

Summary of DNA replication

1. DNA helicase unwind the parental double helix at origin of replication forming bubbles
2. SSB proteins stabilize the unwound parental DNA.
3. leading strand is synthesized continuously in 5'→3' direction by DNA polymerase III
4. lagging strand is synthesized discontinuously. RNA primase synthesizes a short RNA primer, which is extended by DNA polymerase to form okazaki fragments
5. DNA polymerase I removed RNA primer and replaces with DNA
6. DNA joins okazaki fragments together to form a continuous strand.

Properties of chromosomes in nucleus

1. specific chromosomes occupy different regions within the nucleus at different times
2. chromosomes will move from one territory (region where replication takes place)
3. occurs with replication and transcription

Mitochondrial replication

1. DNA separate from the nucleus
2. replicates in G2 phase of cell cycle
3. circular DNA with no assoc. proteins
4. 16,567 bp
5. 13 genes that code for proteins, 22 genes for tRNAs, and 2 genes for rRNAs
6. 5-10 copies per mitochondria (identical copies)
7. 100s-1000s of mitochondria/ cell.

Biotechnology

use of recombinant DNA technology to produce commercial goods and services.

Restriction Enzymes

1. a bacterial enzyme that cuts DNA at specific sites.
2. aka endonucleases

How were restriction enzymes found?

1. discovered by Smith and Nathans
2. bacteria created enzymes that would recognize specific DNA sequences of phage and cut at or near the recognition site.

Naming Restriction enzyme

1. first letter of genus
2. followed by first 2 letters of species
3. then first letter of bacteria strain
4. roman numeral for enzymes that have been discovered by same bacterial species
ie) EcoRI

EcoRI

1. Escheria Coli (strain R, 1st enzyme)
2. Recognition site: G AATTC; CTTAA G
3. staggered cutter

EcoRV

1. Escheria coli (strain R, 5th enzyme)
2. Recognition Site: GAT ATC; CTA TAG
3. Blunt cutter

BamHI

1. Bacillus amyloliquefacians (strain H, enzyme 1)
2. Recognition Site: G GATCC; CCTAG G
3. Staggered cutter

HindIII

1. Haemophilus influenzae (enzyme 3)
2. Recognition Site: A AGCTT; TTCGA A
3. staggered cutter

Staggered Cuts

cuts that produce sticky ends
ie) EcoRI

Blunt Cuts

cuts made right down the middle
ie) EcoRV

Recombinant DNA

1. mix DNA from different sources
2. use same enzyme to make staggered DNA cuts
3. cut DNA from source A and source B, both containing sticky ends that will rejoin and sealed with Ligase making a new piece of DNA
4. combined DNA from source A and source B - new unique sequence of DNA

What uses recombined DNA?

1. unique biological products - proteins that do not exist in nature such as pharmaceuticals, research tools, commercial products
2. important bio-proteins that exist naturally in small quantities
ie) Factor VIII and IX, Insulin

Recombinant DNA Technology

series of techniques in which DNA fragment from an organism are linked to self replicating vectors to create recombinant DNA molecules, which are replicated or cloned in a host cell.

RFLP

1. Restriction Fragment Length Polymorphism
2. genetic test used for : genetic disease testing, paternity testing, forensic evidence, and disease assoc. studies
3. any DNA sequence variation that may create a new restriction site or may delete an existing restriction site
4. restriction enzyme is used to digest DNA and run gel electrophoresis to analyze band sizes.

Where do polymorphic DNA sequences exist in the population?

1. a gene
2. next to a gene
3. in an intron (non-expressed region of a gene)

Southern Blotting

1. method for transferring DNA fragments from a gel to a membrane filter
2. developed by Edwin Southern for use in hybridization experiment.

Southern Blotting is used for...

1. ID and analyze normal and mutant alleles
2. ID related genes in other organisms
3. study genes evolution

Process of Southern Blotting

1. cut DNA - choose restriction enzyme and add to DNA and buffer; incubate at 37°C for several hours
2. electrophoresis - separate different size fragments that are produced by restriction cuts
3. transfer DNA to membrane - soak gel in NaOH to denature DNA, set up transfer apparatus, nylon or nitrocellulose membrane
4. probe membrane - ssDNA probe with complementary sequence to target DNA fragments, radioactive or fluorescent tag (probe at specific temp)
5. analyze bands - gel is transferred via capillary action

Why can't you probe on a gel?

1. gels are too fragile
2. membranes are more durable allow for replicated copies

Problem with Southerns

1. time consuming (1-2 weeks)
2. labor intensive
3. requires high quality and a lot of DNA

Why is Southern blotting necessary?

to amplify specific genes rather than all DNA from genome

How are genes cloned?

1. genes are cloned using plasmids from bacteria
2. bacterial cell will copy it's chromosome and all plasmids passing off all genetic information to daughter cell.
3. use restriction enzymes to cut plasmid and desirable gene.
4. Place desirable gene into plasmid and anneal with ligase.

Vectors

self replicating DNA molecules that are used to transfer foreign DNA segments between host cells.

Types of DNA cloning vectors and their advantages and disadvantages

1. Plasmids - 1-2 kb per plasmid - requires 8 million plasmids to clone entire genome
2. YACS - artificial, linear, yeast, insert upto 1000kb/YAC - requires 3000 YACS to clone entire genome
3. either method is labor intensive, time consuming, and expensive

YACS

1. cloning vector that has telomeres and a centremere that can accumulate large DNA inserts
2. uses the eukaryote, yeast

Probes

1. a labeled nucleic acid to id a complementary region of clone or genome.
2. can base pair with complementary base sequence in gene of interest.

PCR

1. Polymerase Chain Reaction
2. a method for amplifying DNA segments using cycles of denaturation, annealing to primers, DNA polymerase directed toward DNA synthesis.
3. cloning without vectors - making a million copies of specific DNA fragments

Single PCR Cycle

1. denaturation step: heat tube to 90-95°C, allows duplex to unwind by breaking H bonds
2. Annealing step: cool tube to 45-65°C (depends on primer); allows primer to anneal to DNA strands
3. Extension step: heat 70-72°C (optimal for polymerase); allows polymerase to make DNA

PCR results

1. usually done in multiple cycles
2. after n cycles, there is 2ⁿ ↑ in amount of double stranded DNA
3. allows DNA to be replicated in test tube rather than host cell.

PCR Thermocycling

1. used for 1 piece of DNA
2. repeat cycle for 30-40x; allow all 3 steps, usually 30 sec./step
3. amplification becomes logarithmic: in excess of 1 million copies in 30 cycles
4. total time for completion is several hours

Taq Polymerase

1. found in bacteria living in hot springs
2. heat stable polymerase typically used for PCR

Thermocycler

machine does PCR cycling more efficiently

Benefits of PCR

1. quick, easy
2. direct visualization without Southern
3. very little source DNA is needed
4. any sample of DNA can be used such as ancient DNA, highly degraded DNA, blood, cheek swab, hair sample, skin cell, etc

PCR Disadvantages

1. contamination concerns
2. can get non-specific amplification
3. if test failed, then there were procedural issues

VNTRS

1. Variable number of Tandem Repeats
2. 15-1000 bp repeats in the region between genes that a sequence will repeat
3. used in DNA Profiling

STRs

1. Short Tandem Repeats
2. 2-9 bp repeats
3. short nucleotide sequences that are organized through out the genome that are organized into clusters of varying lengths
4. varying number of repeats are analyzed in DNA profiling

Clones

1. genetically identical molecules, cells, or organisms; all derived from a single ancestor.
2. plants were the 1st organism cloned by Charles Steward in the 1950s.

2 methods to clone animals

1. embryo splitting method
2. cell fusion method

Embryo splitting method

1. done in the 1980s
2. used IVF
3. artificially fertilized donor egg
4. embryo develops to 8-16 cell stage
5. cells are separated and grown into many embryos
6. transplant embryos into surrogate.

Cell Fusion Method

1. used IVF
2. all artificially fertilized egg grow to 32 cell stage
3. separate all 32 cells and fuse with enucleated donor ova that develop in 32 embryos
4. transplant 32 embryos to surrogate

Benefits of Cell Fusion method or Embryo Splitting method

cell fusion can use older donor cells.

Nuclear Transfer Method

1. eliminate fusion step
2. inject nucleus from donor animal directly into enucleated donor ova.
3. more efficient version of Cell Fusion Method.

Charles Steward Experiment (1950s)

1. cloned the first organism - carrot
2. broke carrot into several pieces
3. extracted cells by boaring into carrot with a cork screw
4. placed it in a medium to callus for 4-6 weeks
5. transferred calluses (embryos) into a nutrient rich medium to root and develop
6. transferred to soil to continue growth.

Dolly

1. first cloned animal reported in 1997
2. took mammary cells from an adult sheep and reprogrammed it to grow into a sheep.
3. used cell fusion technique

Karry Mullis

1. discovered PCR in 1983 when he was a Cetus employee
2. after reporting discovery, he received a $10,000 bonus and was fired shortly after
3. Cetus put a patent on it and sold it to La Roche for $300,000,000
4. In 1993, Mullis received the Nobel Prize for his discovery of PCR

DNA Profiling

1. study of STR and VNTR allele frequency patterns to identify individuals.
2. typically used in criminal cases, paternity cases, studying human evolution, tracing ancient migrations, and monitoring contamination of food via microorganisms
3. uses statistics, probability theory, and population genetics to estimate frequency of particular profile found in an individual and in the general population.

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