genetics discussion 6 (sager sequencing, forensics) Flashcards
What’s the difference between pre-mRNA and mRNA?
Pre-mRNA = product of transcription
*which is comprised of both EXONS & INTRONS.
mRNA = After transcription pre-mRNA is SPLICED,
*which involves the REMOVE INTRONS
resulting in the formation of mRNA.
What information does Sanger sequencing provide?
tells you the ORDER OF BASE SEQUENCE of DNA
but DOES NOT provide information on
GENE FUNCTION or LOCATION
What are two types of information that Sanger sequencing DOES NOT provide?
the gene FUNCTION
the gene LOCATION
What would happen if only labelled dNTPs were added to the sequencing reaction?
*dNTP = deoxyribonucleotide triphosphate made up of a -phosphate group, a - deoxyribose sugar and a - nitrogenous base. 4 types dNTPs / split into two groups: - purines - pyrimidines.
*The function of dNTPs in PCR
- to expand the growing DNA strand
with the help of
- Taq DNA polymerase.
The dNTPs are bind with the complementary DNA strand by hydrogen bonds.
*The PCR is an in vitro technique of DNA synthesis.
If only labelled dNTPs were added, the sequence wouldn’t elongate as labelled dNTPs signal to terminate the chain.
What would happen if multiple primers were added to the sequencing reaction?
= lots of pieces
and some of those pieces would be the same size.
*A single primer is used so that multiple PCR reactions
ALL START AT THE SAME POINT
and you can build up a picture of the END LETTER when you line up the pieces from SMALL TO LONG,
thus you can then read the sequence.
Why can’t we just look for the ATG codon to find genes?
We can’t just look for ATG Becuase we DON’T KNOW whether it is in place to
- START THE GENE or to simply
- CODE FOR THE AA METHIONINE
to be added to the protein chain.
*A protein chain may have many ATG sequences.
Why can’t genomic DNA alone be used to determine gene sequence?
Genomic DNA will
give BOTH INTRONS & EXONS sequences.
The FINAL GENE SEQUENCE that determines the protein
= is a product of MATURE mRNA
pre-mRNA
= CANNOT DIFFERENTIATE WHICH PART of the sequence is INTRONS & EXONS based on sequence alone
What must be done first before the mRNA sequence can be obtained?
==> “mRNA EXTRACTED FROM TISSUES”
==> then converted to cDNA (copy DNA)
*using REVERSE TRANSCRIPTASE (from viruses)
*bec..we don’t have polymerase to make copies of RNA, we do once we make DNA
==> Then we can work out the mRNA sequence
*as it will be COMPLEMENTARY to the cDNA sequence.
mRNA is first EXTRACTED FROM THE TISSUE
- if mRNA is present==> it means the gene is being transcribed OW transcription factors are in place for transcribing that gene, the only genes being transcribed in that gene are the ones
- specifict to that cell
- important to that cell
What parameters are used to find ORFs in a DNA sequence?
*open reading frames
ORFs must consist of
= a START SEQUENCE “INITIATION SITE” with ATG
= an ending in a frame STOP CODON of either
- TAA
- TGA or
- TAG.
= It also must be found “IN FRAME”.
Then there must be
= SUFFICIENT CODON PRESENT BETWEEN the START codon and the STOP codon to encode for the
- USUAL SIZE OF A GENE.
What are - two reasons why a - disease gene can’t be found by comparing the cDNA sequence of a - diseased and - normal individual?
= only feasible when a single gene is suspected
= NOT POSSIBLE TO SEQUENCE ALL
~18,000 - 30,000 genes
in a diseased and a normal person
= MANY DIFFERENCES WOULD BE FOUND
so you could NOT DETERMINE WH IS RESPONSIBLE
*the diff could be just hair colour phenotype (blonde or brown)
When would a single chromosome look a cross (X)?
= when has been REPLICATED
- It is a DUPLICATED HOMOLOGOUS CHROMOSOME
with
“2 SISTER CHROMATIDS” (X)
Occurs as a result of DNA REPLICATION
- during the cell cycle
When would a single chromosome look like a stick (I)?
= During CELL DIVISION
*the CHROMOSOMES SPLIT and become
“SINGLE SISTER CHROMATIDS” ( I )
*They now look like ‘sticks’ when shown on karyotypes.
How can the two chromosomes in a pair be matched up?
==>STAINED WITH DYE
==>reveals a pattern of lighter and darker BANDS
==> matched by BANDING PATTERN
*Each chromosome has a UNIQUE BANDING PATTERN.
What sort of
- DNA mutations can be seen
in a
“karyotype analysis”?
= LARGE SCALE GENETIC MUTATIONS
eg. trisomy 21 = extra copy of chromosome
EG…
- PARTIAL chromosome DELETIONS
- MISSING chromosomes
- EXTRA COPIES of chromosomes.
Give two examples of chromosomal mutations able to be seen in a karyotype
= Down Syndrome (Trisomy 21)
*an extra chromosome at 21
= Edwards Syndrome (Trisomy 18)
*an extra chromosome at 18
