Genetics lecture 3 Flashcards
1. DNA polymerases and biotechnology 2. Different types of RNA and functions 3.Basics of transcription in bacteria
What is chemically synthesised DNA called
Oligonucleotides
What are synthetic oligonucleotide primers key for development of
- PCR
- DNA sequencing
A pair of oligos can be used to….
Prime the amplification of a target DNA sequence in a cyclical manner.
What are the three steps in the synthesis cycle?
1.Seperate DNA strands by heating to 98 degrees c
2.Reduce the temp to allow oligo primers to anneal.
3.Reduce temp and add dNTPs and DNA polymerase.
Where does the heat-stable DNA polymerase used in PCR come from
Thermophilic bacterium (Themus aquaticus/ Taq DNA pol)
amplication proceeds…
exponentially
what can PCR be used for? Give 2 examples
Diagnostics
Example 1: A chagas outbreak in hometown of Nunez-Mujica inspired him to find a cheap and portable PCR machine that would help diagnose those infected.
Example 2: Rapid Diagnosis rRt -PCR on site diagnosis for animal pathogens eg Portable PCR cycler machines are used to test cattle for TB.
What type of PCR is used for covid testing?
Quantative PCR (qPCR)
What is the major application of PCR
Genotyping of alleles based on allele-specific length differences
Amplification of alleles that show length polymorphisms :
(i) Variable number tandem repeats (VNTRs) such as Microsatellite sequences eg (CA)n
(ii) Widely dispersed throughout the human genome
Explain how PCR can be used for multiplex genotyping
(i) multiplex genotyping uses fluorescent labelled primers designed to amplify alleles of different sizes: these can be separated on the same gel.
(ii) Multiplexing allows alleles at many different loci to be investigated simultaneously.
What are major applications of multiplex genotyping?
1) Forensic science
2)Paternity testing
3)Population genetics
4)Evolutionary genetics
What is DNA sequencing
determining the base sequence of DNA molecules
What method does DNA sequencing use?
“sequencing by synthesis”
Who developed “sequencing by synthesis?
Fred Sanger
what is the key to Sanger sequencing
di-deoxynucleotides
What prevents further elongation go the chain in Sangers sequencing ?
The absence of a 3’O-H group in a di-deoxy nucleotide terminates chain synthesis/ elongation.
what does the primer indicate in sequencing?
The start point
Explain the process of sequencing by synthesis
- Use a radio-labelled primer
2.Add dNTPs and DNA polymerase to extend the primer. - Separate the new DNA strand by size using gel electrophoresis through a polyacrylamide gel.
4.Use auto-radiography to reveal the newly synthesised DNA.
What do fluorescently- labelled dideoxynucleotides do?
Make sequencing more efficient .
What is “shotgun sequencing”?
Sequence millions of small random fragments, computers assemble the sequence.
Benefits of human genomics
(i) to see who will benefit most from a drug
(ii) to identify mutations for disease, disease resistance , drug sensitivity and traits .
Where are genes located?
Nucleus
What is a gene ?
A stretch of DNA that codes for a protein.
Where are proteins synthesised?
In the cytoplasm
How many genes specify one protein?
One
How can DNA in the Nucleus specify the production of proteins in a cytoplasm?
- DNA is located in the nucleus (in chromosomes), but proteins are made in cytoplasm.
2.RNA is synthesised in the nucleus
3.RNA migrates to the cytoplasm where proteins are synthesised.
4.In viral infections: RNA synthesis procedes new viral particle formation.
What is the intermediate used in specifying proteins in cytoplasm?
mRNA
What is the sugar in RNA?
ribose
Name the 4 bases associated with RNA
Adenince
Guanine
Cytosine
Uracil
How does DNA differs from RNA
Single strand vs Double strand
Ribose vs Deoxyribose
Uracil vs Thymine
What is the difference in structure between ribose and deoxyribose?
Ribose has hydroxyl group where deoxyribose only has hydrogen.
Name 4 nucleotides of RNA
AMP
GMP
CMP
UMP
How many major types of RNA do cells contain
3
Name the 3 types of RNA needed for protein synthesis
Messenger RNA, Ribosomal RNA, Transfer RNA.
3 facts about mRNA
1.Encodes information for a specific protein
2.Low abundance
3.Short half-life (rapid turnover)
3 facts about rRNA
1.Most abundant RNA in a cell
2.Very stable
3. Structural and functional component of ribosomes
3 facts about. transfer RNA (tRNA)
1.Very small
2.Key role in translation
3.Abundant and stable
How many types of RNA polymerases are in prokaryotes?
one (RNAP)
How many types of RNA polymerases are in eukaryotes?
3
Which RNA polymerase makes rRNA
RNAP I
Which RNA polymerase makes mRNA
RNAP II
Which RNA polymerase makes tRNA
RNAP III
How does RNA polymerase know where to start and stop making mRNA?
There are signals in the DNA sequence.
Discuss promoters in Prokaryotic transcription
DNA sequences that guide RNAP to the beginning of a gene to start transcription (initiation site)
Discuss terminators in prokaryotic transcription
DNA sequences that specifyy the termination of RNA synthesis (stop) and release of RNAP from the DNA.
What are the 3 steps in RNA synthesis (transcription)
1.Initiation of transcription
2.Elongation
3.Termination
Roles of RNA polymerase
1.Scans DNA to identify promoter
2.Initiates transcription of mRNA
3.Elongates the mRNA chain
4.Terminates transcription
5.Interacts with regulatory proteins : Activators and Repressors.
Discuss 4 steps in binding of polymerase to template DNA.
1.RNA polymerase binds non-specifically to DNA and scans, looking for the promoter.
2.Sigma sub-unit recognises promoter sequence .
3.RNA polymerase and promoter form a “closed promoter complex”
4.RNA polymerase then unwinds about 12 base pairs to form “open promoter complex”.
Discuss 2 mechanisms in termination of transcription in E.Coli.
- Simple: no protein factor required: hairpin structure forms near end of the mRNA which causes RNAP to dissociate.
- Complex (Rho- dependant): helices protein called Rho factor is required to terminate transcription.
