copying DNA and RNA in vitro

explain the diagram in terms of DNA polymerase replication

• DNA polymerase releases a pyrophosphate from deoxyribonucleoside triphosphate (dnTPs) and adds the resulting dnMP to the 3'-hydroxyl end of the primer strand

• Pyrophosphate is cleaved by pyrophosphatase -> reaction is irreversible

in DNA replication, the template strand is read in a (? to ?) direction and the new strand is synthesised in a (? to ?) direction

• template strand: 3’ to 5’ direction

• new strand: 5’ to 3’ direction

what polymerase is used in PCR?

• Taq DNA polymerase

• heat stable → found in bacteria that live in hot springs

PCR process

1. Double stranded DNA needs to be separated into two template single strands

• Denatured by heating

2. Hybridisation -> primers bind to respective sequences on the single stranded template DNA strands

 3. DNA polymerase uses the four dnTPs to synthesise new DNA strands by extending the primers

the hybridisation of the primers in PCR needs a (?) temperature than the denaturing process

cooler (from 95 in denaturing down to 50-60 degrees celsius for hybridisation)

what are the different temperatures for the 3 steps of PCR

1. 95 → to separate the two DNA strands via denaturing

2. 50-60 → allow primers to hybridise to the template strands

3. 72 → optimal temperature for Taq DNA polymerase

each PCR cycle (?) the DNA amount

doubles

components of PCR (list 7)

1. Template DNA -> the double stranded DNA to be amplified

2. Two primers -> specific to each single stranded DNA template

3. Taq DNA polymerase -> heat resistant DNA polymerase that will add dnTPs to primers

4. dnTPs → building blocks for new DNA strand synthesis

5. Buffer -> keeps pH stable

6. Magnesium ions -> required as cofactor for Taq DNA polymerase

7. PCR machine -> provides correct temperatures and timings for each PCR step 

PCR and DNA fragment cloning (explain how they are linked)

1. isolate DNA from cell

2. identify DNA fragment of interest within isolated DNA

3. separate double stranded DNA and add primers

4. PCR amplification → DNA fragments copied

5. new DNA fragments inserted into plasmid

6. plasmid replicates DNA sequence/ synthesises protein (if DNA is a gene sequence

PCR in parental studies - what are short tandem repeats?

• STR’s are repeated base sequences in the human genome

• Individuals inherit specific STR's with a different number of repeats from their mother and from their father

• PCR used to amplify these STR markers found in chromosomes

• Gel electrophoresis is then used to separate the markers and compare between parents and child

explain why father 2 is the child’s biological father

their STR markers are the same -> more match ups than with father 1

PCR in forensic science - explain the diagram

• used to identify crime suspects

• three suspects had three STR loci analysed and amplified using PCR

• the forensic sample (DNA from crime scene) underwent the same PCR amplifications

• gel electrophoresis was then used to compare suspect and sample STRs to identify the perpetrator

does bacterial DNA have introns? how does this impact the expression of eukaryotic genes inserted into bacteria?

• no → bacteria have no introns and therefore no mechanism to remove introns from a primary mRNA

• eukaryotic genes need to undergo splicing to remove non-coding introns

• therefore eukaryotic protein cannot be produced in a bacteria

cDNA

complimentary DNA → mature mRNA converted to DNA to allow for eukaryotic protein synthesis in bacteria

cDNA synthesis process

1. Eukaryotic mRNAs have a poly-A tail which hybridises with a poly(T)primer

2. Reverse transcriptase

• Synthesises DNA in 5' to 3' direction

• Creates a RNA-DNA hybrid

• Found in retroviruses

3. RNaseH

• Degrades RNA in newly synthesised RNA-DNA hybrid

• Leaves some RNA undegraded

• This RNA acts as a primer for complementary DNA or cDNA synthesis by DNA polymerase

reverse transcription - PCR is used to produce a (a) copy of an (b)

(a) DNA

(b) mRNA

RT-PCR explained

1. mRNA isolates from eukaryotic cells

2. Add DNA primer to 3' poly A tail of mRNA

3. Reverse transcriptase adds deoxyribonucleoside triphosphates to synthesise DNA strand

4. RNaseH degrades most of RNA from DNA-RNA hybrid

5. Leaves some RNA which acts as primer for cDNA

6. DNA polymerase adds dnTPs to primer

7. PCR → add PCR primers, Taq DNA polymerase and dNTPs for PCR amplification

8. Creates cDNA clones which can be ligated into plasmid vectors for replication

PCR or RT-PCR can be used to detect the presence of a viral genome in a sample