D1.1

What does the semiconservative process of DNA replication mean? Why is it important?

Each new double stranded DNA has 1 strand OG DNA strand + 1 strand new DNA

Occurs because each strand of OG DNA is a template for new DNA

Allows a high degree in accuracy when coping base sequences and stability in passing down code

Define and summarize PCR and its materials needed

• Def: a technique used to amplify small DNA fragments

• Used to help clone genes, use crime DNA, sequence extinct DNA

• Desired DNA is put into a reaction chamber, involving:

  • free nucleoside triphosphates, primers (single stranded, short polymers of 15-20 nucleotides that are complementary to nucleotides at the target DNA), taq polymerase

  • thermocycler

List the 3 steps in PCR

1. Denaturation

• DNA is heated to break H-bonds (98C)

2. annealing

• temp is lowered (60C) to allow primers to bind to complementary sequences in the DNA sample

3. extension

• taq polymerase replicates DNA using primers as the starting point (72C)

Why are primers used in PCR?

Serves as a starting point for replication since DNA polymerases can’t add the first nucleotide but extend

Summarize gel electrophoresis and its purpose/applications

• Uses electricity to move molecules through a semisolid medium to separate DNA by size + charge

• helps identify key features of DNA

• DNA profiling

List 3 key steps in gel electrophoresis

1. to get proper sizes of DNA, its digested using restriction endonucleases (enzymes)

• this cuts the backbone at specific sequences

2. samples of DNA fragments are loaded into wells at one end of gel

• gel is soaked in buffer solution

3. electric current runs through gel

• DNA moves to the positive side (since DNA is negatively charged)

• Due to the porous gel, smaller and most charged DNA pieces travel further

• Length of sample DNA fragments can be found by comparing it against DNA ladder

What substance is used to see DNA in gel electrophoresis

Ethidium bromide to help it fluoresce

What contributes to directionality of DNA replication?

The fact that phosphodiester bonds (occurring between phosphate group on 5’C and hydroxyl group on 3’C) are holding nucleotides

Which direction is new DNA built? Why?

• From 5’ to 3’

• Since DNA polymerase III has an active site complementary to a specific shape

What is the lagging vs. leading strand? Why does this exist?

Both refer to the NEW DNA strands formed

Leading (the template strand is 3’ to 5’)

• the strand replicated in the same direction as helicase moves

• formed in continuous way (5’ to 3’)

  • thus, primer and primase are only needed once

• formed quickly

Lagging

• Doesn’t allow DNA polymerase III to move in same direction as helicase

• Moves away from replication fork

• is synthesized discontinuously in okazaki fragments, formed slowly

  • Thus, RNA primer needs to be added repeatedly

• requires DNA ligase

This exists due to the antiparallel nature of DNA

List the 6 enzymes of DNA replication

1. Helicase

• unwinds/unzips DNA by breaking H-bonds (moves one complementary base pair at a time)

• forms a replication fork

• Works with single strand binding proteins, preventing the strand from coming back together

2. Gyrase

• Moves ahead of helicase

• relieves the tension created by the unzipping of double helix

3. DNA primase

• attaches RNA primers to the template strand

  • without it: DNA polymerase III can’t attach to DNA strand

• allows DNA polymerase III to later assemble free nucleotides into a new DNA strand

• For leading strand: 1 primer needed

• For Lagging strand: primers need to be placed at regular intervals

4. DNA polymerase III

• places free nucleotides in correct sequence based on complementary base pairing rule

• replicates continuously on leading strand, discontinuously on lagging strand

5. DNA polymerase I

• removes RNA nucleotides of primers and replaces with DNA nucleotides

6. DNA ligase

• forms phosphodiester bonds between okazaki fragments

• Allows the lagging strand to form one single strand

Which enzyme is needed for DNA proofreading?

• DNA polymerase III (its additional function)

• It proofreads newly formed DNA strand as its being built

• it replaces incorrect nucleotides

What is each nucleotide made of

• Deoxyribose (5C sugar)

• phosphate group

• nitrogenous base

What are 2 major molecules found in nucleus important for DNA replication?

• Enzymes

• free nucleotides (freely floating in nucleoplasm, like adenine, thymine, etc)

Describe the steps for continuous and discontinuous replication

Continuous

• DNA primase adds RNA primer

• DNA polymerase III adds nucleotides to 3’ end

• DNA polymerase I replaces primer with nucleotides

discontinuous

• DNA primase adds RNA primer in front of 5’ end

• DNA polymerase III adds nucleotides

• DNA polymerase replaces primer

• DNA ligase attaches Okazaki fragment ot the lagging strand

List purposes of DNA primers in PCR

• primers attach to the separated strands of DNA

• primers prevent hydrogen bonds between the separated bases from reforming OR prevent original helix from recombining

• provide a starting point for replication as Taq polymerase attaches the first nucleotide to the 3'-end of the primer

• primers can be designed to target specific sequences in the genome

Outline how DNA profiling works

a. sample of DNA obtained from person/hair/blood/mouth/crime scene ✔

b. PCR used to amplify/make copies of DNA (in sample) ✔

c. using Taq DNA polymerase / using DNA polymerase from thermophilic bacteria ✔

d. tandem repeats amplified/used ✔

e. gel electrophoresis used to separate DNA (into bands) ✔

f. separation according to length of fragments/number of repeats
OR
fragments of same length/number of repeats travel same distance ✔

g. pattern of bands/numbers of repeats is the profile/is unique to the individual ✔

h. example of application/forensics/crime investigation/paternity ✔

how are tandem repeats used in DNA profiling

1. tandem repeats (at one locus) vary in number of times sequence repeats / represent different alleles for one locus;

2. DNA sample cut by restriction enzymes into fragments;

3. samples of DNA are amplified at specific genetic sites with PCR;

4. the fragments are separated by their size/number of repeats with gel electrophoresis;

5. fluorescent/radioactive label attached to different tandem repeats;

6. data from several loci at one time uniquely identify individuals / like a fingerprint, combinations of alleles are specific to an individual;

7. comparisons/similarities between fragment patterns to determine paternity/evidence match to a suspect’s profile / other example of comparison/similarity;