D1.1 DNA replication

DNA replication definition

DNA replication is the synthesis of new strands of DNA with the same base sequence as the original strands

daughter molecules of DNA replication in organisms

• Organisms replicate all their DNA before cell division → both daughter cells have the entire genome

• Unicellular organisms → reproduce by cell division

use of DNA replication in multicellular organisms

• growth → requires extra body cells to be produced

• repair of damage tissues by replacing cells

• reproduction → provide cells that develop into gametes.

why is DNA replication semi-conservative

each of the DNA molecules produced has one new strand and one stand conserved from the parent molecule

what happens at the start of DNA replication

• separation of parent DNA molecule into 2 strands by breaking hydrogen bonds between bases (done by helicase)

• each parent strand is used as a template for the assembling of new polymer nucleotides.

why is complementary base pairing important in DNA replication

• allows DNA molecules produced to be identical in base sequence.

• ensures high degree of accuracy in copying of base sequences → very rare for the wrong base to be inserted

replication fork definition + role

• = the site where a parent DNA molecule is separated into two single strands.

• the replication fork moves along the parent molecule

• changes that occur on the replication fork are carried out by helicase and DNA polymerase enzymes

DNA replication Stage 1

Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds

DNA replication Stage 2

DNA polymerase links nucleotides together with phosphodiester bonds to form new strands, using the pre-existing strands as templates.

DNA replication Stage 3

The daughter DNA molecules each rewind into a double helix

what is PCR used for

• polymerase chain reaction is used for copying DNA artificially

• it is carried out in small tubes called eppendorfs which are loaded into a thermocycler.

what is Taq DNA polymerase

• a special type of heat stable DNA polymerase

• allows high temperatures to be used, speeding up replication

what is the use of primers in polymerase chain reaction (PCR)

• Primers = short DNA strands that bind to DNA in the sample after it has been split into single strands by heat

• Primers are made with the base sequence needed to bind at the point where DNA polymerase should attach to the DNA and start copying

why are 2 primers needed in PCR

one is needed for each of the two single strands formed when the double-stranded DNA in the sample is split

what is the use of DNA nucleotides in PCR

used to assemble new strands

repeated cycle of temperature changes in PCR

• DNA is heated to 95C to separate the two strands

• temperature is reduced to 53C allowing primers to bind to both strands of DNA

• temperature increases to 73C encouraging Taq DNA polymerase to replicate both strands, starting at the primer, producing two double stranded copies of the original DNA

• this then repeats in a circle

DNA amplification

• the fact that there are twice as many copies of the desired DNA base sequence after each cycle of replication

• exponential growth

Gel electrophoresis definition

the method of separating mixtures of positively or negatively charged macromolecules.

how does Gel electrophoresis work generally

• the mixture is placed at one end of a the thin sheet of gel

• the gel is a mesh of polymers of an inert material with fluid filled spaces in between

• an electric field is applied to the gel by placing electrodes on both ends of the gel

• charged molecules move to their respective electrode

where does DNA move in Gel electrophoresis

• DNA moves towards the positive electrode (anode) as phosphate groups in DNA are negatively charged

• therefore DNA is placed next to the cathode at the start of Gel electrophoresis

How does Gel electrophoresis work for separating DNA

• the mesh of polymers restricts movement → smaller molecules move faster

• Therefore gel electrophoresis separates DNA based on the size of the molecules.

• when the smallest molecules reach the anode the current is switched off

• a stain is used to reveal DNA in the gel

• DNA molecules of the same size form a band in the gel

What are PCR and gel electrophoresis used for

• generate DNA profiles (genetic fingerprints)

• Forensic investigations → whether DNA profile matches DNA from the crime scene

• Paternity test → whether man is father of a child (DNA profiles of mother and child are needed)

how is a DNA profile generated

• A sample of DNA from a person is taken

• Primers are used to promote the amplification of DNA of about 15 different short tandem repeats (SPRs) by the polymerase chain reaction

• the amplified SPRs are separated by gel electrophoresis generating a pattern of bands that is very likely to be unique to the individual