dna replication and pcr ppt
TRANSCRIPT
Human Genetics
DNA Makes RNA Makes Protein
Terminology Review Chromosome
Threadlike structures in the nucleus that carry genetic information
Gene Fundamental unit of heredity Inherited determinant of a phenotype
Locus Position occupied by a gene on a chromosome
Gene sequence of DNA that instructs a cell to produce a particular
protein DNA
Deoxyribonucleic Acid-the molecule that forms genes The genetic material
Allele Different DNA sequences possible for the same gene location
“A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.”
-Francis Crick
DNA (deoxyribonucleic acid) is a chain of nucleotides
Sugar: Deoxyribose Phosphate Base - one of four types: adenine (A), thymine (T)
guanine (G), cytosine (C)
Which of these are Purine bases?Pyrimidine bases?
A (adenine) C (cytosine) T (thymidine) G (guanine)
A guy walks into a bar and says "My name's Chargaff, and 22% of my DNA is "A" nucleotides. I'll bet anyone that they can't guess what percentage of my DNA is "C" nucleotides!" You say "I'm thirsty, so I'll take that bet!" http://escience.ws/b572/L1/L1.htm
DNA Bases Pair through Hydrogen Bonds
Erwin Chargaff observed:
# of adenine = # of thymine
# of guanine = # of cytosine
Complementary bases pair:
A and T pair
C and G pair
Cytosine deamination (i.e. water attacks!)
DEAMINATION--------->
Cytosine Uracil
What's the difference between DNA and RNA?DNA contains the sugar deoxyribose while RNA is made with the sugar ribose. It's just a matter of a single 2' hydroxyl, which deoxyribose doesn't have, and ribose does have. Of course, you all remember that RNA uses the base uracil instead of thymine too.
Cytosine naturally has a high rate of deamination to give uracil
What's the difference between DNA and RNA?
DNA contains the sugar deoxyribose while RNA is made with the sugar ribose. It's just a matter of a single 2' hydroxyl, which deoxyribose doesn't have, and ribose does have.
You all remember that RNA uses the base uracil instead of thymine too.
Cytosine naturally has a high rate of deamination to give uracil
If C-U deamination occurs and then is replicated, the U will pair
with an A not the C with a G
DEAMINATION--------->
Cytosine Uracil
If 5-methyl C-T deamination occurs and then is replicated, the T will pair with an A not the C with a G
DEAMINATION--------->
5 methyl Cytosine Thymine
DNA is a Double Helix
C
T
GA
C
TG
A
C
T
GA
C
AG
C
TGA
C
TG
X-ray diffraction indicated DNA has a repeating structure.
- Maurice Wilkins and Rosalind Franklin
DNA is double-stranded molecules wound in a double helix.
-James Watson and Francis Crick
DNA Double Helix
A sugar and phosphate “backbone” connects nucleotides in a chain.
DNA has directionality. Two nucleotide chains
together wind into a helix Hydrogen bonds between
paired bases hold the two DNA strands together.
DNA strands are antiparallel
PA
P C
PG
P T
P C
PG
PA
PC
PT
GP
PC P
PG
P
5’
3’
3’
5’
Orientation of DNA
The directionality of a DNA strand is due to the orientation of the phosphate-sugar backbone.
The carbon atoms on the sugar ring are numbered for reference. The 5’ and 3’ hydroxyl groups (highlighted on the left) are used to attach phosphate groups.
Structure of DNA Two nucleic acid chains running in opposite directions The two nucleic acid chains are coiled around a central
axis to form a double helix For each chain – the backbone comes from linking the
pentose sugar bases between nucleotides via phosphodiester bonds connecting via 3’ to 5’
The bases face inward and pair in a highly specific fashion with bases in the other chain
A only with T, G only with C Because of this pairing – each strand is complementary
to the other 5’ ACGTC 3’
3’ TGCAG 5’ Thus DNA is double stranded
Chromatin = DNA and associated proteins
DNA winds aroundhistone proteins (nucleosomes).
Other proteins wind DNA into more tightlypacked form, thechromosome.
Unwinding portions of the chromosome isimportant for mitosis,replication and makingRNA.
Genes: molecular definition
A gene is a segment of DNA which directs the formation of RNA which in turn directs formation of a protein
The protein (or functional RNA) creates the phenotype
Information is conveyed by the sequence of the nucleotides
Why is DNA good Genetic Material?
A linear sequence of bases has a high storage capacity
a molecule of n bases has 4n combinations
just 10 nucleotides long -- 410 or 1,048,576 combinations
Humans – 3.2 x 109 nucleotides long – 3 billion base pairs
Required properties of a genetic material
Chromosomal localization
Control protein synthesis
Replication
DNA Replication- the process of making new copies of the DNA molecules
Potential mechanisms:
organization of DNA strands
Conservative old/old + new/new
Semiconservative old/new + new/old
Dispersive mixed old and new on each strand
Meselson and Stahl’s replication experiment
Conclusion: Replication is semiconservative.
Replication as a process
Double-stranded DNA unwinds.
The junction of the unwound
molecules is a replication fork.
A new strand is formed by pairing complementary bases with theold strand.
Two molecules are made.
Each has one new and one old
DNA strand.
Fig 8.14
Replication in vivo is complex
Replication requires the coordinated regulation of many enzymes and processes unwind the DNA synthesize a new nucleic acid polymer proof read repair mistakes
Enzymes in DNA replication
Helicase unwinds parental double helix
Binding proteinsstabilize separatestrands
DNA polymerase binds nucleotides to form new strands
Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone
Primase adds short primer to template strand
Exonuclease removesRNA primer and inserts the correct bases
Binding proteins prevent single strands from rewinding.
Replication
Helicase protein binds to DNA sequences called origins and unwinds DNA strands.
5’ 3’
5’
3’
Primase protein makes a short segment of RNA complementary to the DNA, a primer.
3’ 5’
5’ 3’
Replication
Overall directionof replication
5’ 3’
5’
3’
5’
3’
3’ 5’
DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
DNA polymerases require an underlying template (and a primer) and cannot synthesize in the direction 3' to 5'. That is, they cannot add nucleotides to a free 5' end.
Replication
DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
5’
5’
Overall directionof replication
5’
3’
5’
3’
3’
3’
DNA polymerase proofreads bases added and replaces incorrect nucleotides.
Replication
5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Leading strand synthesis continues in a 5’ to 3’ direction.
Replication
3’ 5’ 5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Okazaki fragment
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
5’
Replication
5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Okazaki fragment
Replication
5’
5’ 3’
5’
3’
3’
5’
3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Replication
3’
5’
3’
5’
5’ 3’
5’
3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Replication
5’
5’
3’ 3’
5’
3’
5’ 3’
5’
3’
3’
5’
Exonuclease enzymes remove RNA primers.
Replication
Exonuclease enzymes remove RNA primers.
Ligase forms bonds between sugar-phosphate backbone.
3’
5’
3’
5’ 3’
5’
3’
3’
5’
Replication
5’ 3’5’
3’
5’
3’
3’ 5’
5’
5’ 3’5’ 3’ 3’
5’
3’
5’ 3’
5’
3’
3’ 5’
5’ 3’
3’ 5’ 5’
5’ 3’5’ 3’ 3’
5’
3’
5’
5’ 3’5’ 3’
3’
5’ 5’ 3’
5’
3’ 3’ 5’
3’
5’ 3’
5’ 3’
3’
5’
3’ 5’
3’
5’ 3’
5’ 3’
3’
5’
General rules of conduct for DNA polymerase I enzymes (like Taq)
1. Remember your base pairing rules: G goes with C and A goes with T.
2. The 5' ends are strictly off limits
3. There will be no synthesis without a free 3' end
4. There will be no degradation without a free 3' end
General rules of conduct for DNA polymerase I enzymes (like Taq)
5. There will be no synthesis without an underlying template
6. Under no circumstances may you make a synthetic addition to the 5‘ end
7. There is no reconstruction of a broken phosphodiester bond, unless you have ligase. If you are synthesizing DNA and run into an obstruction on your template, you must stop and leave the nick unrepaired.
General rules of conduct for DNA polymerase I enzymes (like Taq)
8.If you have been provided with a free 3' end, a template, and a substrate molecule that is correct, you must add that nucleotide to the growing end of the strand (i.e. to the 3' end.)
PCR: Polymerase Chain Reaction
Selective replication and amplification of specific(targeted) DNA sequences.
PCR basics Know some sequence of the piece of genomic or
other DNA to be amplified DNA primers - short DNA pieces of sequences
complementary to the DNA sequence to be amplified
Four nucleotide building blocks Taq1 - DNA polymerase, Buffer, MgCl2
Polymerase Chain Reaction (PCR)
Denaturation
Each DNA primer anneals, bindingto its complementary sequenceon the template DNA
DNA template is melted with high heat to separate strands.
Annealing
Extension DNA polymerase creates a new strand of DNA complementaryto the template DNA starting from the primer’s free 3’ end.
Multiple rounds of denaturation-annealing-extension areperformed to create many copies of the template DNA between the two primer sequences.
Polymerase Chain Reaction (PCR)DNA template is denatured with heat to separate strands.
C T T G A T CGC3’5’
G ATCAA GCG
3’ 5’
DNA template is melted with heat to separate strands.
C T T G A T CGC
3’5’
G ATCAA GCG
3’ 5’
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer.
C T T G A T CGC
3’5’
G ATCAA GCG
3’ 5’
C T T
GCG
5’
5’
3’
C G CG A T
G A A C T A
3’
Polymerase Chain Reaction (PCR)
Template Base Pairing
Requires correct temperature. Too hot and nothing can form hydrogen
bonds. Too cold and the template reforms and
the primers can form weak hydrogen bonds with sequences that are not perfectly complementary.
http://escience.ws/b572/L3/L3.htm
Genome and Epigenome vary in Monozygotic Twins
Identical Twins don’t actually have completely identical DNA
http://www.cell.com/AJHG/abstract/S0002-9297(08)00102-X
Bruder et al. Phenotypically Concordant and Discordant Monozygotic Twins Display Different DNA Copy-Number-Variation Profiles AJHG, Vol 82, No, 3, 763-771
http://www.nytimes.com/2008/03/11/health/11real.html?scp=3&sq=epigenetics&st=cse
