Gene Sequencing Flashcards
What is PCR?
Polymerase Chain Reaction
-A technique for amplification of DNA in vitro
What is the process of PCR?
-A sample of DNA is taken and undergoes PCR
-The DNA is heated to 95 degrees causing the hydrogen bonds to break and separate the two strands of DNA (denaturation)
-The mixture is then cooled to 55 degrees allowing the primers to anneal to the 3’ end of each strand (annealing)
-It is then heated again to 72 degrees allowing Taq polymerase to attach nucleotides
-The process is then repeated several times to amplify the DNA (extension)
Why is Taq polymerase used?
It is heat resistant as it is taken from bacteria living in thermal hot springs
What is a genome?
The entire genetic material in an organism
Who invented PCR?
Kary Mullis in 1993
What is DNA sequencing?
Identifying the base sequence of a DNA fragment
What is the process of DNA sequencing?
-DNA is chopped into smaller pieces and double strands are separated into single strands
-PCR is used to amplify DNA
-A primer is added
-The fragments are added to four test tubes (A, T, G, C) each containing nucleotides and a polymerase enzyme
-The fragments grow until a terminator base stops them
-Chains are denatured and put in gel electrophoresis
What is a terminator base?
Modified versions of bases with a florescent tag attached which stops anymore bases from being added
What happens when DNA has been sequenced?
The raw data is fed into a computer system that reassembles the genome by looking at overlap between fragments
How can we use DNA sequencing?
-Predicting amino acid sequences to help predict which genes code for which proteins and how the amino acids may join in a polypeptide chain
-Disease management by looking at the mutations which cause disease and can see which mutation in the protein causes symptoms
What is the process of DNA profiling?
-DNA sample is cloned using PCR
-DNA polymerase and free nucleotides are added
-DNA is heated to break the hydrogen bonds
-DNA is cooled and primers are added
-DNA polymerase joins free nucleotides together
-Cycle is repeated to clone multiple copies of DNA
-Cloned DNA is cut into fragments of different lengths using different restriction enzymes
-Fragments are placed into wells and gel electrophoresis is carried out
-DNA fragments separate according to size and move towards anode
-Position of fragments is compared with other samples obtained
-Matching bands can be used to identify crime suspects, give a medical diagnosis or for paternity cases
What is an allele?
Different versions of the same gene
-Everyone has the genes for hair, alleles cause different hair colours
What does a transcription factor do?
-Inhibitor blocks the binding site of the transcription factor
-IAA enters the cell and binds to the transcription factor
-Causes inhibitor to be released
-IAA and transcription factor travel from the cytoplasm to the nucleus
-The transcription factor binds to the promoter sequence of DNA to allow transcription
-mRNA is produced which can be used to synthesise proteins
What is a transcription factor?
A protein that binds to the DNA and affects transcription by turning certain genes on or off
What are the two kinds of transcription factors?
-Activators = stimulate or increase the rate of transcription
-Repressors = inhibit or decrease the rate
What is a stem cell?
Unspecialised cells that can divide and develop into other types of cells
What are the types of stem cells?
-Totipotent = can create any cell type
-Pluripotent = can create most cell types
-Multipotent = can only produce a limited number of cells
-Unipotent = can only differentiate into one cell type
What are the early stages of development?
-Cleavage happens which is rapid mitosis without interphase
-Creates a blastocyst which is a mass of small, identical and undifferentiated cells
What is epigenetics?
Studies genetic control by factors other than the base sequences on the DNA
What is embryonic stem cells?
Undifferentiated cells of the early human embryo with the potential to develop into many different types of specialised cells
What is an adult stem cell/somatic stem cell?
Undifferentiated cells found among the normal differentiated cells in a tissue or organ that can differentiate when needed to produce any one of the major cell types
What happens in transcription?
-Occurs in the nucleus
-A gene on the DNA is transcribed into pre-mRNA
-Introns are removed and exons are spliced together to form mRNA
-mRNA leaves the nucleus via a nuclear pore
What happens in splicing?
-Introns are removed from mRNA by enzymes which cut them out
-The exons are then joined together to form the completed mRNA
-mRNA then leaves the nucleus via a nuclear pore and enters the cytoplasm
What is an intron?
Non-coding regions of DNA
What is an exon?
Coding regions of DNA
What is a mini satellite?
10-100 base sequence, repeated 50 to several hundred times
What is micro satellite?
2-6 bases, repeated 5 to 100 times
What is pre-mRNA?
The mRNA that is transcribed directly from the DNA before it has been modified
What is the situation of a satellite?
Same position on each chromosome and is dependant on inheritance
What is therapeutic cloning?
An experimental technique to produce large quantities of healthy tissues
What are the steps of therapeutic cloning?
-Remove the nucleus from a patient’s normal body cell
-Transfer it to a human ovum which has had its nucleus removed
-Fuse them with an electric shock which causes further development to take place
-Pre embryo starts to develop and divide producing embryonic stem cells
-The genetics perfectly match the patient and stem cells can be collected and cultured to differentiate into the tissue needed
-They can be transferred back into the patient to reduce rejection
What are the cons of stem cell therapy?
-Nobody knows how the genes in the cells are switched on or off to form particular types of tissue
-Could cause the development of cancer
What are the positives of induced pluripotent stem cells (iPS cells)?
-No ethical issues
-Removes the risk of rejection
What are the negatives of induced pluripotent stem cells (iPS cells)?
-Difficult to turn cells into pluripotent cells
-Difficult to persuade pluripotent cells to differentiate into tissues wanted
How do we make induced pluripotent stem cell?
-Use a modified virus carrying human genes
-The virus enters the human cells and inserts the genes into the host DNA
-Transcription factors are introduced into the cells which allows them to become pluripotent
