D1.1: DNA Replication

Purpose of DNA Replication

Production of new, identical DNA to the existing DNA

DNA structure allows for repeated replication, without limitations to number of times

Replication Uses (2)

Reproduction - offspring need copies of parental DNA

Growth and tissue replacement - both require cell division and DNA must be replicated beforehand

What allows for high degree of accuracy during replication?

Semi-conservative model and complementary base pairing

Semi-Conservative Model

both original strands serve as templates for the new strands

-possible bc of copmlementary base pairng (A-T and C-G)

-produces DNA we/ one original (parent, template, antisense) strand and one new (daughter, nontemplate, sense) strand

Allows for high degree of accuracy during replication

Parent Strand

The template for constructing the new DNA double helix, AKA the antisense strand

Daughter Strand

the newly made stand in DNA replication

Complementary Base Pairing

Only complementary bases can be added to new strand b/c H-Bonds cannot form between non-complementary strands

*this is how DNA polymerase III recognizes and replaces mispairings*

Directionality of DNA Polymerase

All nucleotides have 2 covalent bonds, except for those at the ends of the strand

add new nucleotides to the 3' end

phosphate groups are at 5' end

**build 5'-->3' and read 3'-->5'

Deoxynucleoside Triphosphate

dNTPs

free floating dNTPs have 3 phosphates, DNA polymerase cleaves 2 phosphates off and uses the energy to form a phosphodiester bond at 3' end of strand

Significance of 3' End

DNA polymerase NEEDS a 3' end to attach dNTPs

Therefore it can extend, but CANNOT initiate replication (needs RNA primer)

Enzymes in DNA Replication (5)

Helicase, DNA Primase, DNA Polymerase III, DNA Polymerase I, DNA Ligase

Helicase

Separates/unwinds DNA by breaking hydrogen bonds btwn nitrogenous bases

This occurs at the origin of replication, creates 2 replication forks (one on either side)

two parental strands act as templates

DNA Primase

lays down short RNA primers (10-15 bp) which provide a 3' end for DNA polymerase III to bind to and add nucleotides

DNA Polymerase III

binds to template strand at 3' end of RNA primer, NEEDS A 3' END, CAN EXTEND BUT CANNOT INITIATE

Covalently bonds complementary DNA nucleotides, growing chain in 5'-->3' direction

Forms phosphodiester bonds between deoxyribose sugar of one nucleotide and phosphate of another

Cleaves two phosphates off dNTPs and uses energy to create bonds

PROOFREADS EACH NUCLEOTIDE

DNA Polymerase I

Functions as an exonuclease and polymerase

Removes RNA primers and replaces them with DNA nucleotides in 5'-->3' direction

*Cannot make 3' to 5' links, leaves a gap where sugar-phosphate bond would be*

Exonuclease

an enzyme that removes successive nucleotides from the end of a polynucleotide molecule

DNA Ligase

Covalently bonds sugar-phosphate backbone to form a continuous strand

JOINS 3' end to 5' of next nucleotide (the gap that DNA Polymerase I can't form bonds

Joins okazaki fragments

Leading Strand

Makes DNA continuously, grows TOWARDS replication fork (5'-->3')

Completed more quickly, needs RNA primer only ONCE

Lagging Strand

Makes DNA discontinuously, grows away from replication fork

*Has to backtrack to add nucleotides sequentially as replication fork moves away and exposes more of template strand*

Produces short DNA fragments (okazaki fragments), needs RNA primers every 100-200 bps

Okazaki Fragments

Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand.

Leading vs Lagging Strand

Leading: elongate continuously into the widening replication fork

Lagging: replicates away from the fork, must wait until it widens to polymerize and is discontinuous, leading to Okazaki fragments

DNA Proofreading

Occurs immediately after wrong base has been added

Once recognized, DNA Polymerase III excises (removes) incorrect base, moves back one nucleotide and inserts correct base

Polymerase Chain Reaction (PCR)

Automated method of DNA replication, generates large amounts of specific DNA in short period of time, requires small quantity initially

*doubles about every 2-3 mins*

PCR Materials

1. DNA of interest (about 100 bps)

2. DNA primers

3. free nucleosides (dNTPs)

4. Taq polymerse

Taq polymerse

derived from a hot spring bacteria (Thermus aquaticus)

Can function in high temp. w/out denaturing

PCR Process

1. Denature

2. Anneal

3. Elongate

Denature

1st step in PCR at about 95 C, separates DNA strands by breaking H-bonds

Anneal

2nd step in PCR at about 55 C, allows primers to bind

Elongate

3rd step in PCR at about 72 C, optimal temperature for Taq polymerse, assembles new DNA strands

Gel Electrophoresis

Separates DNA fragments according to length

-place samples in "wells" in agar gel

-negatively charged phosphates of DNA travel towards ANODE (positive electrode of power source)

Shorter strands have less resistance to gel and therefore travel farther

Differing sizes separate into bands as they travel at different speeds

Dye in Gel Electrophoresis

Gel is treated w/ dye to make bands (DNA) visible

Banding Pattern

Determined by DNA sequence and type of restriction enzyme used

Size of DNA fragment is determined by comparing it to a know marker (ladder)

Restriction Enzyme

Enzyme that cuts DNA at a specific sequence of nucleotides

Satellite DNA

non-coding regions or "junk DNA"

Contains short tandem repeats (STRs) of DNA that are repeated a varied number of times (ex. TAGA or AGAA)

Short Tandem Repeats

Short sequences of DNA (2-7 bp) repeated a varied number of times

People vary in number and location, producing unique DNA profile (banding pattern)

13 or more STRs are used in DNA profiling (12 different STR locations in chromosome(

Excised (cut) using restriction enzymes and separated via Gel Electrophoresis

What are Gel Electrophoresis bands made of?

Each STR is cut into pieces (ex. for STR: AGAA --> atcgatcgatcgA BREAK GAAatcgatcgatcg)

This cuts the junk DNA into pieces w/ the STRs dividing them, and therefore different sections of junk DNA are different lengths bc STRs located randomly within Junk DNA

The different lengths of these junk DNA sections result in the banding pattern

**thickness of bands kinda irrelevant**

DNA Profiling Procedure

1. Collect DNA (From blood/semen)

2. amplify via PCR

3. cut all samples using same restriction enzyme (different STRS will generate different fragment lengths

4. Separate via Gel Electrophoresis

5. Compare profiles

DNA Profiling Uses (2)

Forensic Investigation and Paternity Testing

Forensic Investigation

to convict, suspect's DNA needs to have a 100% match w/ the DNA from crime scene

Population size in area is considered when determining the uniqueness of profile (ex. smaller population = more unique profiles = less STRs used)

Paternity Testing

children inherit their chromosomes from both parents

All the child's DNA must match either that of mother or father

*to find father, must be the man w/ banding pattern that matches everything that mom doesn't cover w/ her banding pattern for the kid*

Reason for Paternity Testing (2)

Men denying paternity to avoid financial responsibility

Children proving paternity for inheritance rights